Method for identifying lead compounds

ABSTRACT

The present invention is a method for ranking the affinity of each of a multiplicity of different molecules for a target molecule which is capable of denaturing due to a thermal change. The method comprises contacting the target molecule with one molecule of the multiplicity of different molecules in each of a multiplicity of containers, simultaneously heating the multiplicity of containers, measuring in each of the containers a physical change associated with the thermal denaturation of the target molecule resulting from the heating in each of the containers, generating a thermal denaturation curve for the target molecule as a function of temperature for each of the containers and determining a midpoint temperature (T m ) therefrom, comparing the T m  of each of the thermal denaturation curves with the T m  of a thermal denaturation curve obtained for the target molecule in the absence of any of the molecules in the multiplicity of different molecules, and ranking the affinities of the multiplicity of different molecules according the change in T m  of each of the thermal denaturation curves.

This application is a divisional of U.S. application Ser. No. 08/853,464filed May 9, 1997 (U.S. Pat. No. 6,020,141), which claims prioritybenefit of U.S. provisional application No. 60/017,860, filed May 9,1996, the entirety of which is incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant nos. 1 R43GM52786-01 and 2 R44 GM52786-02, awarded by National Institute ForGeneral Medical Research. The U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the screening of compound andcombinatorial libraries. More particularly, the present inventionrelates to a method and apparatus for performing assays, particularlythermal shift assays.

2. Related Art

In recent years, pharmaceutical researchers have turned to combinatoriallibraries as sources of new lead compounds for drug discovery. Acombinatorial library is a collection of chemical compounds which havebeen generated, by either chemical synthesis or biological synthesis, bycombining a number of chemical “building blocks” as reagents. Forexample, a combinatorial polypeptide library is formed by combining aset of amino acids in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can theoretically be synthesized through suchcombinatorial mixing of chemical building blocks. Indeed, oneinvestigator has observed that the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gordon, E. M. et al., J. Med. Chem. 37:1233-1251 (1994)).

The rate of combinatorial library synthesis is accelerated by automatingcompound synthesis and evaluation. For example, DirectedDiversity® is acomputer based, iterative process for generating chemical entities withdefined physical, chemical and/or bioactive properties. TheDirectedDiversity® system is disclosed in U.S. Pat. No. 5,463,564, whichis herein incorporated by reference in its entirety.

Once a library has been constructed, it must be screened to identifycompounds which possess some kind of biological or pharmacologicalactivity. To screen a library of compounds, each compound in the libraryis equilibrated with a target molecule of interest, such as an enzyme. Avariety of approaches have been used to screen combinatorial librariesfor lead compounds. For example, in an encoded library, each compound ina chemical combinatorial library can be made so that an oligonucleotide“tag” is linked to it. A careful record is kept of the nucleic acid tagsequence for each compound. A compound which exerts an effect on thetarget enzyme is selected by amplifying its nucleic acid tag using thepolymerase chain reaction (PCR). From the sequence of the tag, one canidentify the compound (Brenner, S. et al., Proc. Natl. Acad. Sci. USA89:5381-5383 (1992)). This approach, however, is very time consumingbecause it requires multiple rounds of oligonucleotide tag amplificationand subsequent electrophoresis of the amplification products.

A filamentous phage display peptide library can be screened for bindingto a biotinylated antibody, receptor or other binding protein. The boundphage is used to infect bacterial cells and the displayed determinant(i.e., the peptide ligand) is then identified (Scott, J. K. et al.,Science 249:386-390 (1990)). This approach suffers from severaldrawbacks. It is time consuming. Peptides which are toxic to the phageor to the bacterium cannot be studied. Moreover, the researcher islimited to investigating peptide compounds.

In International Patent Application WO 94/05394 (1994), Hudson, D. etal., disclose a method and apparatus for synthesizing and screening acombinatorial library of biopolymers on a solid-phase plate, in an arrayof 4×4 to 400×400. The library can be screened using a fluorescentlylabeled, radiolabeled, or enzyme-linked target molecule or receptor. Thedrawback to this approach is that the target molecule must be labeledbefore it can be used to screen the library.

A challenge presented by currently available combinatorial libraryscreening technologies is that they provide no information about therelative binding affinities of different ligands for a receptor protein.This is true whether the process for generating a combinatorial libraryinvolves phage library display of peptides (Scott, J. K. et al., Science249:386-390 (1990)), random synthetic peptide arrays (Lam, K. S. et al.,Nature 354:82-84 (1991)), encoded chemical libraries (Brenner, S. etal., Proc. Natl. Acad Sci. USA 89:5381-5383 (1992)), the method ofHudson (Intl. Appl. WO 94/05394), or most recently, combinatorialorganic synthesis (Gordon, E. et al., J. Med. Chem 37:1385-1399 (1994)).

To acquire quantitative binding data from the high throughput screeningof ligand affinities for a target enzyme, researchers have relied onassays of enzyme activity. Enzymes lend themselves to high throughputscreening because the effect of ligand binding can be monitored usingkinetic assays. The experimental endpoint is usually aspectrophotometric change. Using a kinetic assay, most researchers use atwo-step approach to lead compound discovery. First, a large library ofcompounds is screened against the target enzyme to determine if any ofthe library compounds are active. These assays are usually performed ina single concentration (between 10⁻⁴-10⁻⁶ M) with one to threereplicates. Second, promising compounds obtained from the first screen(i.e., compounds which display activity greater than a predeterminedvalue) are usually re-tested to determine a 50% inhibitory concentration(IC₅₀), an inhibitor association constant (K_(i)), or a dissociationconstant (K_(d)). This two-step approach, however, is very laborintensive, time-consuming and prone to error. Each re-tested sample musteither be retrieved from the original assay plate or weighed out andsolubilized again. A concentration curve must then be created for eachsample and a separate set of assay plates must be created for eachassay.

There are other problems associated with the biochemical approach tohigh throughput screening of combinatorial libraries. Typically, a givenassay is not applicable to more than one receptor. That is, when a newreceptor becomes available for testing, a new assay must be developed.For many receptors, reliable assays are simply not available. Even if anassay does exist, it may not lend itself to automation. Further, if aK_(i) is the endpoint to be measured in a kinetic assay, one must firstguess at the concentration of inhibitor to use, perform the assay, andthen perform additional assays using at least six differentconcentrations of inhibitor. If one guesses too low, an inhibitor willnot exert its inhibitory effect at the suboptimal concentration tested.

In addition to the drawbacks to the kinetic screening approach describedabove, it is difficult to use the kinetic approach to identify and rankligands that bind outside of the active site of the enzyme. Sinceligands that bind outside of the active site do not prevent binding ofspectrophotometric substrates, there is no spectrophotometric change tobe monitored. An even more serious drawback to the kinetic screeningapproach is that non-enzyme receptors cannot be assayed at all.

Thermal protein unfolding, or thermal “shift,” assays have been used todetermine whether a given ligand binds to a target receptor protein In aphysical thermal shift assay, a change in a biophysical parameter of aprotein is monitored as a function of increasing temperature. Forexample, in calorimetric studies, the physical parameter measured is thechange in heat capacity as a protein undergoes temperature inducedunfolding transitions. Differential scanning calorimetry has been usedto measure the affinity of a panel of azobenzene ligands forstreptavidin (Weber, P. et al., J. Am. Chem. Soc. 16:2717-2724 (1994)).Titration calorimetry has been used to determine the binding constant ofa ligand for a target protein (Brandts, J. et al., American Laboratory22:30-41 (1990)). The calorimetric approach, however, requires that theresearcher have access to a calorimetric device. In addition,calorimetric technologies do not lend themselves to the high throughputscreening of combinatorial libraries, three thermal scans per day areroutine.

Like calorimetric technologies, spectral technologies have been used tomonitor temperature induced protein unfolding (Bouvier, M. et at.,Science 265:398-402 (1994); Chavan, A. J. et al, Biochemistry33:7193-7202 (1994); Morton, A. et al., Biochemistry 1995:8564-8575(1995)). The single sample heating and assay configuration, asconventionally performed, has impeded the application of thermal shifttechnologies to high throughput screening of combinatorial libraries.Thus, there is a need for a thermal shift technology which can be usedto screen combinatorial libraries, can be used to identify and rank leadcompounds, and is applicable to all receptor proteins.

Thermal shift assays have been used to determine whether a ligand bindsto DNA. Calorimetric, absorbance, circular dichroism, and fluorescencetechnologies have been used (Pilch, D. S. et al., Proc. Natl. Acad Sci.U.S.A 91:9332-9336 (1994); Lee, M. et al., J. Med. Chem. 36:863-870(1993); Butour, J.-L. et al., Eur. J. Biochem. 202:975-980 (1991);Barcelo, F. et al., Chem. Biol Interactions 74:315-324 (1990)). As usedconventionally, however, these technologies have impeded the highthroughput screening of nucleic acid receptors for lead compounds whichbind with high affinity. Thus, there is a need for a thermal shifttechnology which can be used to identify and rank the affinities of leadcompounds which bind to DNA sequences of interest.

When bacterial cells are used to overexpress exogenous proteins, therecombinant protein is often sequestered in bacterial cell inclusionbodies. For the recombinant protein to be useful, it must be purifiedfrom the inclusion bodies. During the purification process, therecombinant protein is denatured and must then be renatured. It isimpossible to predict the renaturation conditions that will facilitateand optimize proper refolding of a given recombinant protein. Usually, anumber of renaturing conditions must be tried before a satisfactory setof conditions is discovered. In a study by Tachibana et al., each offour disulfide bonds were singly removed, by site-directed mutagenesis,from hen lysozyme (Tachibana et al., Biochemistry 33:15008-15016(1994)). The mutant genes were expressed in bacterial cells and therecombinant proteins were isolated from inclusion bodies. Each of theisolated proteins were renatured under different temperatures andglycerol concentrations. The efficacy of protein refolding was assessedin a bacteriolytic assay in which bacteriolytic activity was measured asa function of renaturing temperature. The thermal stability of eachprotein was studied using a physical thermal shift assay. In this study,however, only one sample reaction was heated and assayed at a time. Thesingle sample heating and assay configuration prevents the applicationof thermal shift technologies to high throughput screening of amultiplicity of protein refolding conditions. Thus, there is a need fora thermal shift technology which can be used to rank the efficacies ofvarious protein refolding conditions.

Over the past four decades, X-ray crystallography and the resultingatomic models of proteins and nucleic acids have contributed greatly toan understanding of structural, molecular, and chemical aspects ofbiological phenomena. However, crystallographic analysis remainsdifficult because there are not straightforward methodologies forobtaining X-ray quality protein crystals. Conventional methods cannot beused quickly to identify crystallization conditions that have highestprobability of promoting crystallization (Garavito, R. M. et al., J.Bioenergtics and Biomembranes 28:13-27 (1996)). Even the use offactorial design experiments and successive automated grid searches(Cox, M. J., & Weber, P. C., J. Appl. Cryst. 20:366-373 (1987); Cox, M.J., & Weber, P. C., J. Crystal Growth 90:318-324 (1988)) do notfacilitate rapid, high throughput screening of biochemical conditionsthat promote the crystallization of X-ray quality protein crystals.Moreover, different proteins are expected to require differentconditions for protein crystallization, just as has been the experiencefor their folding (McPherson, A., In: Preparation and Analysis ofProtein Crystals, Wiley lnterscience, New York, (1982)). Conventionalmethods of determining crystallization conditions are cumbersome, slow,and labor intensive. Thus, there is a need for a rapid, high throughputtechnology which can be used to rank the efficacies of proteincrystallization conditions.

Rapid, high throughput screening of combinatorial molecules orbiochemical conditions that stabilize target proteins in thermal shiftassays would be facilitated by the simultaneous heating of many samples.To date, however, thermal shift assays have not been performed that way.Instead, the conventional approach to performing thermal shift assayshas been to heat and assay only one sample at a time. That is,researchers conventionally 1) heat a sample to a desired temperature ina heating apparatus; 2) assay a physical change, such as absorption oflight or change in secondary, tertiary, or quaternary protein structure;3) heat the samples to the next highest desired temperature; 4) assayfor a physical change; and 5) continue this process repeatedly until thesample has been assayed at the highest desired temperature.

This conventional approach is disadvantageous for at least two reasons.First, this approach is labor intensive. Second, this approach limitsthe speed with which thermal shift screening assays can be performed andthereby precludes rapid, high-throughput screening of combinatorialmolecules binding to a target receptor and biochemical conditions thatstabilize target proteins. Thus, there is a need for an apparatuscapable of performing rapid, high-throughput thermal shift assays thatwill be suitable for all receptors, including reversibly foldingproteins.

SUMMARY OF THE INVENTION

The present invention provides a multi-variable method for ranking theefficacy of one or more of a multiplicity of different molecules ordifferent biochemical conditions for stabilizing a target molecule whichis capable of denaturing due to a thermal change. The method comprisescontacting the target molecule with one or more of a multiplicity ofdifferent molecules or different biochemical conditions in each of amultiplicity of containers, simultaneously heating the multiplicity ofcontainers, measuring in each of the containers a physical changeassociated with the thermal denaturation of the target moleculeresulting from heating, generating a thermal denaturation curve for thetarget molecule as a function of temperature for each of the containers,comparing each of the denaturation curves to (i) each of the otherthermal denaturation curves and to (ii) the thermal denaturation curveobtained for the target molecule under a reference set of biochemicalconditions, and ranking the efficacies of multiplicity of differentmolecules or the different biochemical conditions according to thechange in each of the thermal denaturation curves.

The present invention provides a multi-variable method for optimizingthe shelf life of a target molecule which is capable of denaturing dueto a thermal change. The method comprises contacting the target moleculewith one or more of a multiplicity of different molecules or differentbiochemical conditions in each of a multiplicity of containers,simultaneously heating the multiplicity of containers, measuring in eachof the containers a physical change associated with the thermaldenaturation of the target molecule resulting from heating, generating athermal denaturation curve for the target molecule as a function oftemperature for each of the containers, comparing each of thedenaturation curves to (i) each of the other thermal denaturation curvesand to (ii) the thermal denaturation curve obtained for the target undera reference set of biochemical conditions, and ranking the efficacies ofmultiplicity of different molecules or the different biochemicalconditions according to the change in each of the thermal denaturationcurves.

The present invention also provides a multi-variable method for rankingthe affinity of a combination of two or more of a multiplicity ofdifferent molecules for a target molecule which is capable of denaturingdue to a thermal change. The method comprises contacting the targetmolecule with a combination of two or more different molecules of themultiplicity of different molecules in each of a multiplicity ofcontainers, simultaneously heating the multiplicity of containers,measuring in each of the containers a physical change associated withthe thermal denaturation of the target molecule resulting from theheating, generating a thermal denaturation curve for the target moleculeas a function of temperature for each of the containers, comparing eachof the thermal denaturation curves with (i) each of the other thermaldenaturation curves obtained for the target molecule and to (ii) thethermal denaturation curve for the target molecule in the absence of anyof the two or more different molecules, and ranking the affinities ofthe combinations of the two or more multiplicity of different moleculesaccording to the change in each of the thermal denaturation curves.

The present invention also provides a multi-variable method for rankingthe efficacies of one or more of a multiplicity of different biochemicalconditions to facilitate the refolding of a sample of a denaturedprotein. The method comprises placing one of the refolded proteinsamples in each of a multiplicity of containers, wherein each of therefolded protein samples has been previously refolded according to oneor more of the multiplicity of conditions, simultaneously heating themultiplicity of containers, measuring in each of the containers aphysical change associated with the thermal denaturation of the proteinresulting from heating, generating a thermal denaturation curve for theprotein as a function of temperature for each of the containers,comparing each of the denaturation curves to (i) each of the otherthermal denaturation curves and to (ii) the thermal denaturation curveobtained for the native protein under a reference set of biochemicalconditions, and ranking the efficacies of the multiplicity of differentrefolding conditions according to the change in the magnitude of thephysical change of each of the thermal denaturation curves.

The present invention also provides a further multi-variable method forranking the efficacies of one or more of a multiplicity of differentbiochemical conditions to facilitate the refolding of a sample of adenatured protein, which comprises determining one or more combinationsof a multiplicity of different conditions which promote proteinstability, folding the denatured protein under said one or morecombinations of biochemical conditions that were identified as promotingprotein stabilization, assessing folded protein yield, ranking theefficacies of said multiplicity of different refolding conditionsaccording to folded protein yield, and repeating these steps until acombination of biochemical conditions that promote optimal proteinfolding are identified.

Using the microplate thermal shift assay, one can determine one or morebiochemical conditions have an additive effect on protein stability.Once a set of biochemical conditions that facilitate an increase inprotein stability have been identified using the thermal shift assay,the same set of conditions can be used in protein folding experimentswith recombinant protein. If the conditions that promote proteinstability in the thermal shift assay correlate with conditions thatpromote folding of recombinant protein, conditions can be furtheroptimized by performing additional thermal shift assays until acombination of stabilizing conditions that result in further increaseprotein stability are identified. Recombinant protein is then foldedunder those conditions. This process is repeated until optimal foldingconditions are identified.

The present invention also provides a multi-variable method for rankingthe efficacy of one or more of a multiplicity of different biochemicalconditions for facilitating the crystallization on of a protein which iscapable of denaturing due to a thermal change. The method comprisescontacting the protein with one or more of the multiplicity of differentbiochemical conditions in each of a multiplicity of containers,simultaneously heating the multiplicity of containers, measuring in eachof the containers a physical change associated with the thermaldenaturation of the protein resulting from the heating, generating athermal denaturation curve for the protein as a function of temperaturefor each of the containers, comparing each of the denaturation curves to(i) each of the other thermal denaturation curves and (ii) to thethermal denaturation curve obtained using a reference set of biochemicalconditions, and ranking the efficacies of the multiplicity of differentbiochemical conditions according to the change in each of the thermaldenaturation curves.

The present invention also provides a method for ranking the affinity ofeach of a multiplicity of different molecules for a target moleculewhich is capable of denaturing due to a thermal change. The methodcomprises contacting the target molecule with one molecule of amultiplicity of different molecules in each of a multiplicity ofcontainers, simultaneously heating the containers, measuring in each ofthe containers a physical change associated with the thermaldenaturation of the target molecule resulting from heating, generating athermal denaturation curve for the target molecule as a function oftemperature in each of the containers, comparing each of the thermal deration curves with the thermal denaturation curve obtained for thetarget molecule in the absence of any of the molecules in themultiplicity of different molecules, and ranking the affinities of eachmolecule according to the change in each of the thermal denaturationcurves.

The present invention also provides a method for assaying a pool orcollection of a multiplicity of different molecules for a molecule whichbinds to a target molecule which is capable of denaturing due to athermal change. The method comprises contacting the target molecule witha collection of at least two molecules of a multiplicity of differentmolecules in each of a multiplicity of containers, simultaneouslyheating the multiplicity of containers, measuring in each of thecontainers a physical change associated with the thermal denaturation ofthe target molecule resulting from heating, generating a set of thermaldenaturation curves for the target molecule as a function of temperaturefor each of the containers, comparing each of the thermal denaturationcurves with the thermal denaturation curve obtained for the targetmolecule in the absence of any of the molecules in the multiplicity ofdifferent molecules, ranking the affinities of the collections ofdifferent molecules according to the change in each of the thermaldenaturation curves, selecting the collection of different moleculeswhich contains a molecule with affinity for the target molecule,dividing the selected collection into smaller collections of moleculesin each of a multiplicity of containers, and repeating the above stepsuntil a single molecule responsible for the original thermal shift inthe multiplicity of molecules is identified.

This invention also provides an improved method for generating leadcompounds which comprises synthesizing a multiplicity of compounds andtesting the ability of each compound to bind to a receptor molecule. Theimprovement comprises contacting the receptor molecule with one compoundof a multiplicity of different compounds in each of a multiplicity ofwells in a microplate, simultaneously heating the wells, measuring ineach of the wells a physical change, resulting from heating, associatedwith the thermal denaturation of the receptor molecule, generating athermal denaturation curve for the receptor molecule as a function oftemperature in each of the wells, comparing each of the thermaldenaturation curves with the thermal denaturation curve obtained for thereceptor molecule in the absence of any of the compounds in themultiplicity of different compounds, and ranking the affinities of eachcompound according to the change in each of the thermal denaturationcurves.

The present invention also provides a product of manufacture whichcomprises a carrier having a multiplicity of containers therein, each ofthe containers containing a target molecule which is capable ofdenaturation due to heating, and at least one molecule selected from amultiplicity of different molecules present in a combinatorial library,wherein each of the different molecules are present in a different oneof the multiplicity of containers in the carrier.

Optimization of protein stability, ligand binding, protein folding, andprotein crystallization are multi-variable events. Multi-variableoptimization problems require large numbers of parallel experiments tocollect as much data as possible in order to determine which variablesinfluence a favorable response. For example, multi-variable optimizationproblems require large numbers of parallel experiments to collect asmuch data as possible in order to determine which variables influenceprotein stability. In this regard, both protein crystallization andquantitative structure activity relationship analyses have greatlybenefited from mass screening protocols that employ matrix arrays ofincremental changes in biochemical or chemical composition. Thus, inmuch the same way that quantitative structure activity relationships areconstructed to relate variations of chemical functional groups onligands to their effect on binding affinity to a given therapeuticreceptor, the methods and apparatus of the present invention facilitatethe construction of a quantitative model that relates differentbiochemical conditions to experimentally measured protein stability,ligand specificity, folded protein yield, and crystallized proteinyield.

The present invention offers a number of advantages over previoustechnologies that are employed to optimize multi-variable events such asprotein stabilization, ligand binding, protein folding, and proteincrystallization. Foremost among these advantages is that the presentinvention facilitates high throughput screening. Further, the presentinvention offers a number of advantages over previous technologies thatare employed to screen combinatorial libraries. Foremost among theseadvantages is that the present invention facilitates high throughputscreening of combinatorial libraries for lead compounds. Many currentlibrary screening technologies simply indicate whether a ligand binds toa receptor or not. In that case, no quantitative information isprovided. No information about the relative binding affinities of aseries of ligands is provided. In contrast, the present inventionfacilitates the ranking of a series of compounds for their relativeaffinities for a target receptor. With this information in hand, astructure-activity relationship can be developed for a set of compounds.The ease, reproducibility, and speed of using ligand-dependent changesin midpoint unfolding temperature (T_(m)) to rank relative bindingaffinities makes the present invention a powerful tool in the drugdiscovery process.

Typically, the conventional kinetic screening approach requires at leastsix additional well assays at six different concentrations of inhibitorto determine a K_(i). Using the present invention, throughput isenhanced ˜6 fold over the enzyme based assays because one completebinding experiment can be performed in each well of a multiwellmicroplate. The kinetic screening approached are even further limited bythe usual compromise between dilution and signal detection, whichusually occurs at a protein concentration of about 1 nM. In this regard,the calorimetric approaches, either differential scanning calorimetry orisothermal titrating calorimetry, are at an even worse disadvantagesince they are limited to solitary binding experiments, usually 1 perhour. In contrast the present invention affords a wide dynamic range ofmeasurable binding affinities, from ˜10⁻⁴ to 10⁻¹⁵ M, in a single well.

The present invention does not require radioactively labeled compounds.Nor does it require that receptors be labeled with a fluorescent orchromophoric label.

A very important advantage of the present invention is that it can beapplied universally to any receptor that is a drug target. Thus, it isnot necessary to invent a new assay every time a new receptor becomesavailable for testing. When the receptor under study is an enzyme,researchers can determine the rank order of affinity of a series ofcompounds more quickly and more easily than they can using conventionalkinetic methods. In addition, researchers can detect ligand binding toan enzyme, regardless of whether binding occurs at the active site, atan allosteric cofactor binding site, or at a receptor subunit interface.The present invention is equally applicable to non-enzyme receptors,such as proteins and nucleic acids.

In a further aspect of the present invention, an assay apparatus isprovided that includes a heating means for simultaneously heating aplurality of samples, and a receiving means for receiving spectralemission from the samples while the samples are being heated. In yet afurther aspect of the present invention, an assay apparatus is providedthat includes a temperature adjusting means for simultaneously adjustinga temperature of a plurality of samples in accordance with apre-determined temperature profile, and a receiving means for receivingspectral emission from the samples while the temperature of the samplesis adjusted in accordance with the temperature profile.

In yet a further aspect, the present invention also provides an assayapparatus that includes a movable platform on which are disposed aplurality of heat conducting blocks. The temperature of the heatconducting blocks, and their samples, are adjusted by a temperatureadjusting means. Each of the plurality of heat conducting blocks isadapted to receive a plurality of samples. A light source is providedfor emitting an excitatory wavelength of light for the samples. Whilethe temperature of the samples is being adjusted, a sensor detects thespectral emission from the samples in response to the excitatorywavelength of light. The movable platform is moved between heatconducting blocks to sequentially detect spectral emission from thesamples in each of the plurality of heat conducting blocks.

The assay apparatus of the present invention affords the artisan theopportunity to rapidly screen molecules and biochemical conditions thataffect protein stability. Samples are simultaneously heated over a rangeof temperatures. During heating, spectral emissions are received. Theassay apparatus of the present invention also provides the artisan withan opportunity for conveniently and efficiently carrying out the methodsof the present invention. The assay apparatus of the present inventionis particularly adapted for carrying out thermal shift assays ofmolecules and biochemical conditions that stabilize target proteins.

Because the apparatus of the present invention comprises both a heatingmeans and a spectral emission receiving means, the apparatus of thepresent invention obviates the need to heat samples in one apparatus andtransfer the samples to another apparatus prior to taking spectralemission readings. As a result, the apparatus of the present inventionfacilitates changing temperature in accordance with a pre-determinedtemperature profile, rather than incremental temperature increases andintermediate cooling steps. Thus, more data points can be collected fora given sample and more accurate information can be obtained.

Further, because the assay apparatus of the present invention comprisesboth a heating means and a spectral emission receiving means, spectralmeasurements can be taken from the samples while they are being heated.Thus, using the assay apparatus of the present invention, the artisancan study both irreversibly unfolding proteins and reversibly foldingproteins.

Further features and advantages of the present invention are describedin detail below with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is described with reference to the accompanyingdrawings. In the drawings, like reference numbers indicate identical orfunctionally similar elements. Additionally, the left-most digit(s) of areference number identifies the drawing in which the reference numberfirst appears.

FIG. 1 shows the results of a microplate thermal shift assay for ligandswhich bind to the active site of human α-thrombin (with turbidity as theexperimental signal).

FIG. 2 shows the results of a microplate thermal shift assay for ligandswhich bind to acidic fibroblast growth factor (aFGF (with turbidity asthe experimental signal).

FIG. 3 shows the results of a microplate thermal shift assay for ligandbinding to the active site of human α-thrombin (with fluorescenceemission as the experimental signal). The lines drawn through the datapoints represent non-linear least squares curve fits of the data usingthe equation shown at the bottom of the figure. There are five fittingparameters for this equation of y(T) vs. T: (1) y_(f), thepre-transitional fluorescence for the native protein; (2) y_(u), thepost-transitional fluorescence for the unfolded protein; (3) T_(m), thetemperature at the midpoint for the unfolding transition; (4) ΔH_(u),the van't Hoff unfolding enthalpy change; and (5) ΔC_(pu), the change inheat capacity upon protein unfolding. The non-linear least squares curvefitting was accomplished using KALEIDAGRAPH™ 3.0 software (SynergySoftware, Reading Pa.), which allows the five fitting parameters tofloat while utilizing Marquardt methods for the minimization of the sumof the squared residuals.

FIG. 4 shows the result of a microplate thermal shift assay of ligandswhich bind to the D(II) domain of human FGF receptor 1 (D(II) FGFR1)(with fluorescence emission as the experimental signal). The lines drawnthrough the data points represent non-linear least squares curve fits ofthe data using the equation shown at the bottom of the figure, asdescribed for FIG. 3.

FIG. 5 shows the results of a miniaturized microplate thermal shiftassay for Factor D in the absence of any ligands.

FIG. 6 shows the results of a microplate thermal shift assay for FactorXa in the absence of any ligands.

FIG. 7 shows the results of a miniaturized microplate thermal shiftassay of a ligand that binds to the catalytic site of human α-thrombin.

FIG. 8 shows the results of a miniaturized microplate thermal shiftassay of aprosulate binding to the D(II) domain of human FGF receptor 1.

FIG. 9 shows the results of a miniaturized microplate thermal shiftassay for urokinase in the presence of glu-gly-arg chloromethylketone.

FIG. 10 shows the results of a miniaturized microplate thermal shiftassay of human α-thrombin in which the assay volume is 2 μl. Thermaldenaturation curves for three experiments are shown.

FIG. 11 shows the results of a miniaturized microplate thermal shiftassay of human α-thrombin in which the assay volume is 5 μl. Thermaldenaturation curves for five experiments are shown.

FIG. 12 shows the results of a single temperature microplate thermalshift assay of human α-thrombin in the presence of four differentcompounds in four separate experiments.

FIG. 13 shows the results of a microplate thermal shift assay of theintrinsic tryptophan fluorescence of human α-thrombin. In this assay,blank well fluorescence was not subtracted from sample fluorescence.

FIG. 14 shows the results of a microplate thermal shift assay of theintrinsic tryptophan fluorescence of human α-thrombin. In this assay,blank well fluorescence was subtracted from sample fluorescence.

FIG. 15 shows the results of microplate thermal shift assays of singleligand binding interactions to three different classes of binding sitesfor human α-thrombin.

FIG. 16 shows the results of microplate thermal shift assays ofmulti-ligand binding interactions for human α-thrombin.

FIGS. 17A-D show the results of microplate thermal shift assays of theeffect of pH and various sodium chloride concentrations on the stabilityof human α-thrombin. In FIG. 17A, the fluorophore is 1,8-ANS. In FIG.17B, the fluorophore is 2,6-ANS. In FIG. 17C, the fluorophore is2,6-TNS. In FIG. 17D, the fluorophore is bis-ANS.

FIG. 18 shows the results of microplate thermal shift assays of theeffect of calcium chloride, ethylenediaminetetraacetic acid,dithiothreitol, and glycerol on the stability of human α-thrombin.

FIG. 19 shows the results of microplate thermal shift assays of theeffect of pH and sodium chloride concentration of the stability of humanD(II) FGF receptor 1.

FIG. 20 shows the results of microplate thermal shift assays of theeffect of various biochemical conditions on the stability of human D(II)FGF receptor 1.

FIG. 21 shows the results of microplate thermal shift assays of theeffect of various biochemical conditions on the stability of human D(II)FGF receptor 1.

FIG. 22 shows the results of microplate thermal shift assays of theeffect of various biochemical conditions on the stability of human D(II)FGF receptor 1.

FIG. 23 shows the results of microplate thermal shift assays of theeffect of various biochemical conditions on the stability of human D(II)FGF receptor 1.

FIG. 24 shows the results of microplate thermal shift assays of theeffect of various biochemical conditions on the stability of human D(II)FGF receptor 1.

FIG. 25 shows the results of microplate thermal shift assays of theeffect of various biochemical conditions on the stability of humanurokinase.

FIG. 26 is a schematic diagram of a thermodynamic model for the linkageof the free energies of protein folding and ligand binding.

FIG. 27 is a schematic diagram of a method of screening biochemicalconditions that optimize protein folding.

FIG. 28 shows the results of microplate thermal shift assays of humanα-thrombin stability using various fluorophores.

FIG. 29 shows a schematic diagram of one embodiment of an assayapparatus of the present invention.

FIG. 30 shows a schematic diagram of an alternate embodiment of theassay apparatus of the present invention

FIG. 31 shows a schematic diagram of the assay apparatus according toanother embodiment of the present invention.

FIGS. 32A-E illustrate one embodiment of a thermal electric stage forthe assay apparatus of the present invention. FIG. 32A shows a side viewof the thermal electric stage. FIG. 32B shows a top view of the thermalelectric stage. FIGS. 32C-E show three configurations of inserts thatcan be attached to the thermal electric stage. In one embodiment,inserts accommodate a microtitre plate. In such an embodiment, assaysamples are contained within the wells of the microtitre plate.

FIG. 33 is a schematic diagram illustrating a top view of anotherembodiment of the assay apparatus of the present invention.

FIG. 34 is a schematic diagram illustrating the top view of theembodiment of the assay apparatus shown in FIG. 33 with a housinginstalled.

FIG. 35 is a schematic diagram illustrating a side view of theembodiment of the assay apparatus shown in FIGS. 33 and 34.

FIGS. 36A and 36B illustrate a temperature profile and how thetemperature profile is implemented using the automated assay apparatusof the present invention.

FIG. 37 shows an exemplary computer system suitable for use with thepresent invention.

FIG. 38 shows a flow diagram illustrating one embodiment forimplementation of the present invention.

FIG. 39 shows a flow diagram illustrating an alternate embodiment forimplementation of the present invention.

FIG. 40 shows a comparison of the results of microplate thermal shiftassays of human α-thrombin denaturation performed using a fluorescencescanner and a CCD camera.

FIGS. 41A and 41B show photographs of microplate thermal shift assay ofhuman α-thrombin denaturation performed using a CCD camera. FIG. 41A:V-bottom well microplate. FIG. 41B: dimple microplate.

FIG. 42 shows a comparison of the results of microplate thermal shiftassays of human α-thrombin denaturation performed using a fluorescencescanner and a CCD camera.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description, reference will be made to various termsand methodologies known to those of skill in the biochemical andpharmacological arts. Publications and other materials setting forthsuch known terms and methodologies are incorporated herein by referencein their entireties as though set forth in full.

Overview of the Methods of the Present Invention

The present invention provides a method for ranking a multiplicity ofdifferent molecules in the order of their ability to bind to a targetmolecule which is capable of unfolding due to a thermal change. In oneembodiment of this method, the target molecule is contacted with onemolecule of a multiplicity of different molecules in each of amultiplicity of containers. The containers are then simultaneouslyheated, in intervals, over a range of temperatures. After each heatinginterval, a physical change associated with the thermal denaturation ofthe target molecule is measured. In an alternate embodiment of thismethod, the containers are heated in a continuous fashion. A thermaldenaturation curve is plotted as a function of temperature for thetarget molecule in each of the containers. Preferably, the temperaturemidpoint, T_(m), of each thermal denaturation curve is identified and isthen compared to the T_(m) of the thermal denaturation curve obtainedfor the target molecule in the absence of any of the molecules in thecontainers. Alternatively, an entire thermal denaturation curve can becompared to other entire thermal denaturation curves using computeranalytical tools.

The term “combinatorial library” refers to a plurality of molecules orcompounds which are formed by combining, in every possible way for agiven compound length, a set of chemical or biochemical building blockswhich may or may not be related in structure. Alternatively, the termcan refer to a plurality of chemical or biochemical compounds which areformed by selectively combining a particular set of chemical buildingblocks. Combinatorial libraries can be constructed according to methodsfamiliar to those skilled in the art. For example, see Rapoport et al.,Immunology Today 16:43-49 (1995); Sepetov, N. F. et al., Proc. Natl.Acad. Sci. U.S.A. 92:5426-5430 (1995); Gallop, M. A. et al., J. Med.Chem. 9:1233-1251 (1994); Gordon, E. M. et al., J. Med. Chem.37:1385-1401 (1994); Stankova, M. et al., Peptide Res. 7:292-298 (1994);Erb, E. et al., Proc. Natl. Acad. Sci. U.S.A. 91:11422-11426 (1994);DeWitt, S. H. et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909-6913 (1993);Barbas, C. F. et al., Proc. Natl. Acad. Sci. U.S.A. 89:4457-4461 (1992);Brenner, S. et al. Proc. Natl. Acad. Sci. U.S.A. 89:5381-5383 (1992);Lam, K. S. et al., Nature 354:82-84 (1991); Devlin, J. J. et al.,Science 245:404-406 (1990); Cwirla, S. E. et al., Proc. Natl. Acad. Sci.U.S.A. 87:6378-6382 (1990); Scott, J. K. et al., Science 249:386-390(1990). Preferably, the term “combinatorial library” refers to adirected diversity chemical library, as set forth in U.S. Pat. No.5,463,564. Regardless of the manner in which a combinatorial library isconstructed, each molecule or compound in the library is catalogued forfuture reference.

The term “compound library” refers to a plurality of molecules orcompounds which were not formed using the combinatorial approach ofcombining chemical or biochemical building blocks. Instead, a compoundlibrary is a plurality of molecules or compounds which are accumulatedand are stored for use in future ligand-receptor binding assays. Eachmolecule or compound in the compound library is catalogued for futurereference.

The terms “multiplicity of molecules,” “multiplicity of compounds,” or“multiplicity of containers” refer to at least two molecules, compounds,or containers.

The term “multi-variable” refers to more than one experimental variable.

The term “screening” refers to the testing of a multiplicity ofmolecules or compounds for their ability to bind to a target moleculewhich is capable of denaturing.

The term “ranking” refers to the ordering of the affinities of amultiplicity of molecules or compounds for a target molecule, accordingto the ability of the molecule or compound to shift the thermaldenaturation curve of the target molecule, relative to the thermaldenaturation curve of the target molecule in the absence of any moleculeor compound.

The term “ranking” also refers to the ordering of the efficacies of amultiplicity of biochemical conditions in optimizing proteinstabilization, protein folding, protein crystallization, or proteinshelf life. In the context of optimization of protein stabilization,optimization of protein folding, optimization of proteincrystallization, and optimization of protein shelf life, the term“ranking” refers to the ordering of the efficacies of one or morecombinations of biochemical conditions to shift the thermal denaturationcurve of the target molecule, relative to the thermal denaturation curveof the target molecule under a reference set of conditions.

The term “reference set of conditions” refers to a set of biochemicalconditions under which a thermal denaturation curve for a targetmolecule is obtained. Thermal denaturation curves obtained underconditions different than the reference conditions are compared to eachother and to the thermal denaturation curve obtained for the targetmolecule under reference conditions.

As discussed above, ranking molecules, compounds, or biochemicalconditions according to a change in the T_(m) of a thermal denaturationcurve is preferable. Alternatively; molecules, compounds, or biochemicalconditions can be ranked for their ability to stabilize a targetmolecule according to the change in entire thermal denaturation curve.

The term “lead molecule” refers to a molecule or compound, from acombinatorial library, which displays relatively high affinity for atarget molecule. The terms “lead compound” and “lead molecule” aresynonymous. The term “relatively high affinity” relates to affinities inthe K_(d) range of from 10⁻⁴ to 10⁻¹⁵ M.

The term “target molecule” encompasses peptides, proteins, nucleicacids, and other receptors. The term encompasses both enzymes andproteins which are not enzymes. The term encompasses monomeric andmultimeric proteins. Multimeric proteins may be homomeric orheteromeric. The term encompasses nucleic acids comprising at least twonucleotides, such as oligonucleotides. Nucleic acids can besingle-stranded, double-stranded or triple-stranded. The termencompasses a nucleic acid which is a synthetic oligonucleotide, aportion of a recombinant DNA molecule, or a portion of chromosomal DNA.The term target molecule also encompasses portions of peptides,proteins, and other receptors which are capable of acquiring secondary,tertiary, or quaternary structure through folding, coiling or twisting.The target molecule may be substituted with substituents including, butnot limited to, cofactors, coenzymes, prosthetic groups, lipids,oligosaccharides, or phosphate groups. The term “capable of denaturing”refers to the loss of secondary, tertiary, or quaternary structurethrough unfolding, uncoiling, or untwisting. The terms “target molecule”and “receptor” are synonymous.

Examples of target molecules are included, but not limited to thosedisclosed in Faisst, S. et al., Nucleic Acids Research 20:3-26 (1992);Pimentel, E., Handbook of Growth Factors, Volumes I-III, CRC Press,(1994); Gilman, A. G. et al., The Pharmacological Basis of Therapeutics,Pergamon Press (1990); Lewin, B., Genes V, Oxford University Press(1994); Roitt, I., Essential Immunology, Blackwell Scientific Publ.(1994); Shimizu, Y., Lymphocyte Adhesion Molecules, RG Landes (1993);Hyams, J. S. et al., Microtubules, Wiley-Liss (1995); Montreuil, J. etal., Glycoproteins, Elsevier (1995); Woolley, P., Lipases: TheirStructure Biochemistry and Applications, Cambridge University Press(1994); Kurjan, J., Signal Transduction: Prokaryotic and SimpleEukaryotic Systems, Academic Press (1993); Kreis, T., et al., Guide Bookto the Extra Cellular Matrix and Adhesion Proteins, Oxford UniversityPress (1993); Schlesinger, M. J., Lipid Modifications of Proteins, CRCPress (1992); Conn, P. M., Receptors: Model Systems and SpecificReceptors, Oxford University Press (1993); Lauffenberger, D. A. et al.,Receptors: Models For Binding Trafficking and Signaling, OxfordUniversity Press (1993); Webb, E. C., Enzyme Nomenclature, AcademicPress (1992); Parker, M. G., Nuclear Hormone Receptors; MolecularMechanisms, Cellular Functions Clinical Abnormalities, Academic PressLtd. (1991); Woodgett, J. P, Protein Kinases, Oxford University Press(1995); Balch, W. E. et al., Methods in Enzymology, 257, Pt. C: SmallGTPases and Their Regulators: Proteins Involved in Transport, AcademicPress (1995); The Chaperonins, Academic Press (1996); Pelech, L.,Protein Kinase Circuitry in Cell Cycle Control, RG Landes (1996);Atkinson, Regulatory Proteins of the Complement System, Franklin Press(1992); Cooke, D. T. et al., Transport and Receptor Proteins of PlantMembranes: Molecular Structure and Function, Plenum Press (1992);Schumaker, V. N., Advances in Protein Chemistry: Lipoproteins,Apolipoproteins, and Lipases, Academic Press (1994); Brann, M.,Molecular Biology of G-Protein-Coupled Receptors: Applications ofMolecular Genetics to Pharmacology, Birkhauser (1992); Konig, W.,Peptide and Protein Hormones: Structure, Regulations, Activity—AReference Manual, VCH Publ. (1992); Tuboi, S. et al., Post-TranslationalModification of Proteins, CRC Press (1992); Heilmeyer, L. M., CellularRegulation by Protein Phosphorylation, Springer-Verlag (1991); Takada,Y., Integrin: The Biological Problem, CRC Press (1994); Ludlow, J. W.,Tumor Suppressors: Involvement in Human Disease, Viral ProteinInteractions, and Growth Regulation, RG Landes (1994); Schlesinger, M.J., Lipid Modification of Proteins, CRC Press (1992); Nitsch, R. M.,Alzheimer's Disease: Amyloid Precursor Proteins, Signal Transduction,and Neuronal Transplantation, New York Academy of Sciences (1993);Cochrane, C. G. et al., Cellular and Molecular Mechanisms ofInflammation, Vol. 3: Signal Transduction in Inflammatory Cells, Part A,Academic Press (1992); Gupta, S. et al., Mechanisms of LymphocyteActivation and Immune Regulation IV: Cellular Communications, PlenumPress (1992); Authi, K. S. et al., Mechanisms of Platelet Activation andControl, Plenum Press (1994); Grunicke, H., Signal TransductionMechanisms in Cancer, RG Landes (1995); Latchman, D. S., EukaryoticTranscription Factors, Academic Press (1995).

The term “target molecule” refers more specifically to proteins involvedin the blood coagulation cascade, fibroblast growth factors, andfibroblast growth factor receptors, urokinase, and factor D.

The term “molecule” refers to the compound which is tested for bindingaffinity for the target molecule. This term encompasses chemicalcompounds of any structure, including, but not limited to nucleic acidsand peptides. More specifically, the term “molecule” encompassescompounds in a compound or a combinatorial library. The terms “molecule”and “ligand” are synonymous.

The terms “thermal change” and “physical change” encompass the releaseof energy in the form of light or heat, the absorption of energy in theform or light or heat, changes in turbidity and changes in the polarproperties of light. Specifically, the terms refer to fluorescentemission, fluorescent energy transfer, absorption of ultraviolet orvisible light, changes in the polarization properties of light, changesin the polarization properties of fluorescent emission, changes inturbidity, and changes in enzyme activity. Fluorescence emission can beintrinsic to a protein or can be due to a fluorescence reporter molecule(below). For a nucleic acid, fluorescence can be due to ethidiumbromide, which is an intercalating agent. Alternatively, the nucleicacid can be labeled with a fluorophore (below).

The term “contacting a target molecule” refers broadly to placing thetarget molecule in solution with the molecule to be screened forbinding. Less broadly, contacting refers to the turning, swirling,shaking or vibrating of a solution of the target molecule and themolecule to be screened for binding. More specifically, contactingrefers to the mixing of the target molecule with the molecule to betested for binding. Mixing can be accomplished, for example, by repeateduptake and discharge through a pipette tip. Preferably, contactingrefers to the equilibration of binding between the target molecule andthe molecule to be tested for binding. Contacting can occur in thecontainer (infra) or before the target molecule and the molecule to bescreened are placed in the container.

The target molecule may be contacted with a nucleic acid prior to beingcontacted with the molecule to be screened for binding. The targetmolecule may be complexed with a peptide prior to being contacted withthe molecule to be screened for binding. The target molecule may bephosphorylated or dephosphorylated prior to being contacted with themolecule to be screened for binding.

A carbohydrate moiety may be added to the target molecule before thetarget molecule is contacted with the molecule to be screened forbinding. Alternatively, a carbohydrate moiety may be removed from thetarget molecule before the target molecule is contacted with themolecule to be screened for binding.

The term “container” refers to any vessel or chamber in which thereceptor and molecule to be tested for binding can be placed. The term“container” encompasses reaction tubes (e.g., test tubes, microtubes,vials, etc.). Preferably, the term “container” refers to a well in amultiwell microplate or microtiter plate. The term “sample” refers tothe contents of a container.

A “thermal denaturation curve” is a plot of the physical changeassociated with the denaturation of a protein or a nucleic acid as afunction of temperature. See, for example, Davidson et al., NatureStructure Biology 2:859 (1995); Clegg, R. M. et al., Proc. Natl. AcadSci. U.S.A. 90:2994-2998 (1993).

The “midpoint temperature, T_(m)” is the temperature midpoint of athermal denaturation curve. The T_(m) can be readily determined usingmethods well known to those skilled in the art. See, for example, Weber,P. C. et al., J. Am. Chem. Soc. 116:2717-2724 (1994); Clegg, R. M. etal., Proc. Natl. Acad. Sci. U.S.A. 90:2994-2998 (1993).

The term “fluorescence probe molecule” refers to a fluorophore, which isa fluorescent molecule or a compound which is capable of binding to anunfolded or denatured receptor and, after excitement by light of adefined wavelength, emits fluorescent energy. The term fluorescenceprobe molecule encompasses all fluorophores. More specifically, forproteins, the term encompasses fluorophores such as thioinosine, andN-ethenoadenosine, formycin, dansyl derivatives, fluoresceinderivatives, 6-propionyl-2-dimethylamino)-napthalene (PRODAN),2-anilinonaphtalene, and N-arylamino-naphthalene sulfonate derivativessuch as 1-anilinonaphtalene-8-sulfonate (1,8-ANS),2-anilinonaphthalene-6-sulfonate (2,6-ANS),2-aminonaphthalene6-sulfonate,N,N-dimethyl-2-aminonaphthalene-6-sulfonate,N-phenyl-2-aminonaphthalene,N-cyclohexyl-2-aminonaphthalene-6-sulfonate,N-phenyl-2-aminonaphthalene-6-sulfonate,N-phenyl-N-methyl-2-aminonaph-thalene-6-sulfonate,N-(o-toluyl)-2-aminonaphthalene-6-sulfonate,N-(m-toluyl)-2-aminonaphthalene-6-sulfonate,N-(p-toluyl)-2-aminonaphthalene-6-sulfonate,2-(p-toluidinyl)-naphthalene-6-sulfonic acid (2,6-TNS), 4-(dicyanovinyl)julolidine (DCVJ), 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN),6-hexadecanoyl-2-(((2-trimethylammonium-ethyl)methyl)amino)naphthalenechloride(PATMAN),nile red, N-phenyl-1-naphthylamine, 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP),4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS), andDAPOXYL™ derivatives (Molecular Probes, Eugene, OR). Preferably forproteins, the term refers to 1,8-ANS or 2,6-TNS.

A double-stranded oligonucleotide may be used in fluorescence resonanceenergy transfer assays. One strand of the oligonucleotide will containthe donor fluorophore. The other strand of the oligonucleotide willcontain the acceptor fluorophore. For a nucleic acid to “contain” adonor or an acceptor fluorophore, the fluorophore can be incorporateddirectly into the oligonucleotide sequence. Alternatively, thefluorophore can be attached to either the 5′- or 3′-terminus of theoligonucleotide.

A donor fluorophore is one which, when excited by light, will emitfluorescent energy. The energy emitted by the donor fluorophore isabsorbed by the acceptor fluorophore. The term “donor fluorophore”encompasses all fluorophores including, but not limited to,carboxyfluorescein, iodoacetamidofluorescein, and fluoresceinisothiocyanate. The term “acceptor fluorophore” encompasses allfluorophores including, but not limited to, iodoacetamidoeosin andtetramethylrhodamine.

The term “carrier” encompasses a platform or other object, of any shape,which itself is capable of supporting at least two containers. Thecarrier can be made of any material, including, but not limited toglass, plastic, or metal. Preferably, the carrier is a multiwellmicroplate. The terms microplate and microtiter plate are synonymous.The carrier can be removed from the heating element. In the presentinvention, a plurality of carriers are used. Each carrier holds aplurality of containers.

The term “biochemical conditions” encompasses any component of aphysical, chemical, or biochemical reaction. Specifically, the termrefers to conditions of temperature, pressure, protein concentration,pH, ionic strength, salt concentration, time, electric current,potential difference, concentrations of cofactor, coenzyme, oxidizingagents, reducing agents, detergents, metal ion, ligands, or glycerol.

The term “denatured protein” refers to a protein which has been treatedto remove secondary, tertiary, or quaternary structure. The term “nativeprotein” refers to a protein which possesses the degree of secondary,tertiary or quaternary structure that provides the protein with fullchemical and biological function. A native protein is one which has notbeen heated and has not been treated with denaturation agents orchemicals such as urea.

The term “denatured nucleic acid” refers to a nucleic acid which hasbeen treated to remove folded, coiled, or twisted structure.Denaturation of a triple-stranded nucleic acid complex is complete whenthe third strand has been removed from the two complementary strands.Denaturation of a double-stranded DNA is complete when the base pairingbetween the two complementary strands has been interrupted and hasresulted in single-stranded DNA molecules that have assumed a randomform. Denaturation of single-stranded RNA is complete whenintramolecular hydrogen bonds have been interrupted and the RNA hasassumed a random, non-hydrogen bonded form.

The terms “folding,” “refolding,” and “renaturing” refer to theacquisition of the correct secondary, tertiary, or quaternary structure,of a protein or a nucleic acid, which affords the full chemical andbiological function of the biomolecule.

The term “efficacy” refers to the effectiveness of a particular set ofbiochemical conditions in facilitating the refolding or renaturation ofan unfolded or denatured protein.

The terms “spectral measurement” and “spectrophotometric measurement”refer to measurements of changes in the absorption of light. Turbiditymeasurements, measurements of visible light absorption, and measurementof ultraviolet light absorption are examples of spectral measurements.

The term “polarimetric measurement” relates to measurements of changesin the polarization properties of light and fluorescent emission.Circular dichroism and optical rotation are examples of polarizationproperties of light which can be measured polarimetrically. Measurementsof circular dichroism and optical rotation are taken using aspectropolarimeter. “Nonpolarimetric” measurements are those that arenot obtained using a spectropolarimeter.

The term “collection” refers to a pool or a group of at least onemolecule to be tested for binding to a target molecule or receptor.

A “host”is a bacterial cell that has been transformed with recombinantDNA for the purpose of expressing protein which is heterologous to thehost bacterial cell.

The thermal shift assay is based on the ligand-dependent change in thethermal denaturation curve of a receptor, such as a protein or a nucleicacid. When heated over a range of temperatures, a receptor will unfold.By plotting the degree of denaturation as a function of temperature, oneobtains a thermal denaturation curve for the receptor. A useful point ofreference in the thermal denaturation curve is the temperature midpoint(T_(m)), the temperature at which the receptor is half denatured.

Ligand binding stabilizes the receptor (Schellman, J., Biopolymers14:999-1018 (1975)). The extent of binding and the free energy ofinteraction follow parallel courses as a function of ligandconcentration (Schellman, J., Biophysical Chemistry 45:273-279 (1993);Barcelo, F. et al., Chem. Biol. Interactions 74:315-324 (1990)). As aresult of stabilization by ligand, more energy (heat) is required tounfold the receptor. Thus, ligand binding shifts the thermaldenaturation curve. This property can be exploited to determine whethera ligand binds to a receptor: a change, or “shift”, in the thermaldenaturation curve, and thus in the T_(m), suggests that a ligand bindsto the receptor.

The thermodynamic basis for the thermal shift assay has been describedby Schellman, J. A. (Biopolymers 15:999-1000 (1976)), and also byBrandts et al. (Biochemistry 29:6927-6940 (1990)). Differential scanningcalorimetry studies by Brandts et al., (Biochemistry 29:6927-6940(1990)) have shown that for tight binding systems of 1:1 stoichiometry,in which there is one unfolding transition, one can estimate the bindingaffinity at T_(m) from the following expression: $\begin{matrix}{K_{L}^{T_{m}} = \frac{\begin{matrix}{\exp \left\{ {{- {\frac{\Delta \quad H_{u}^{T_{0}}}{R}\left\lbrack {\frac{1}{T_{m}} - \frac{1}{T_{0}}} \right\rbrack}} +} \right.} \\\left. {\frac{\Delta \quad C_{pu}}{R}\left\lbrack {{\ln \left( \frac{T_{m}}{T_{0}} \right)} + \frac{T_{0}}{T_{m}} - 1} \right\rbrack} \right\}\end{matrix}}{\left\lbrack L_{T_{m}} \right\rbrack}} & \left( {{equation}\quad 1} \right)\end{matrix}$

where

K_(L) ^(T) ^(_(m)) =the ligand association constant at T_(m);

T_(m)=the midpoint for the protein unfolding transition in the presenceof ligand;

T₀=the midpoint for the unfolding transition in the absence of ligand;

ΔH_(u) ^(T) ^(₀) =the enthalpy of protein unfolding in the absence ofligand at T₀;

ΔC_(pu) ^(u)=the change in heat capacity upon protein unfolding in theabsence of ligand;

[L_(T) _(m) ]=the free ligand concentration at T_(m); and

R=the gas constant.

The parameters ΔH_(u) and ΔC_(pu) are usually observed from differentialscanning calorimetry experiments and are specific for each receptor. Tocalculate the binding constant from equation 1, one should have accessto a differential scanning calorimetry instrument to measure ΔH_(u) andΔC_(pu) for the receptor of interest. One can also locate theseparameters for the receptor of interest, or a receptor closely relatedto it, in the literature. In these situations, equation (1) will allowthe accurate measurement of K_(L) at T_(m).

It is also possible to calculate the ligand association constant at anytemperature, K_(L) at T, using equation 2. To use equation 2,calorimetry data for the binding enthalpy at T, ΔH_(L), and the changeof heat capacity upon ligand binding, ΔC_(pL) must be known (Brandts etal., Biochemistry 29:6927-6940 (1990)). $\begin{matrix}{K_{L}^{T} = {K_{L}^{T_{m}}\exp \left\{ {{- {\frac{\Delta \quad H_{L}^{T}}{R}\left\lbrack {\frac{1}{T} - \frac{1}{T_{m}}} \right\rbrack}} + {\frac{\Delta \quad C_{pL}}{R}\left\lbrack {{\ln \left( \frac{T}{T_{m}} \right)} - \frac{T}{T_{m}} + 1} \right\rbrack}} \right\}}} & \left( {{equation}\quad 2} \right)\end{matrix}$

where

K_(L) ^(T)=the ligand association constant at any temperature T;

K_(L) ^(T) ^(_(m)) =the ligand association constant at T_(m);

T_(m)=the midpoint for the protein unfolding transition in the presenceof ligand;

ΔH_(L) ^(T)=the enthalpy of ligand binding in the absence of ligand atT;

ΔC_(pL)=the change in heat capacity upon binding of ligand; and

R=the gas constant.

The second exponential term of equation 2 is usually small enough to beignored so that approximate values of K_(L) at T can be obtained usingjust the first exponential term: $\begin{matrix}{K_{L}^{T} = {K_{L}^{T_{m}}\exp \left\{ {- {\frac{\Delta \quad H_{L}^{T}}{R}\left\lbrack {\frac{1}{T} - \frac{1}{T_{m}}} \right\rbrack}} \right\}}} & \left( {{equation}\quad 3} \right)\end{matrix}$

One need not, however, calculate binding constants according toequations 1-3 in order to rank the affinities of a multiplicity ofdifferent ligands for a receptor. Rather, the present invention providesa method for ranking affinities of ligands according to the degree towhich the thermal denaturation curve is shifted by the ligand Thus, itis possible to obtain estimates of K_(L) at T_(m), even in the absenceof accurate values of ΔH_(u), ΔC_(pu), and ΔH_(L).

The present invention is particularly useful for screening acombinatorial or a compound library. To achieve high throughputscreening, it is best to house samples on a multicontainer carrier orplatform. A multicontainer carrier facilitates the heating of aplurality of samples simultaneously. In one embodiment, a multiwellmicroplate, for example a 96 or a 384 well microplate, which canaccommodate 96 or 384 different samples, is used as the carrier.

In one embodiment, one sample is contained in each well of a multi-wellmicroplate. The control well contains receptor, but no molecule to betested for binding. Each of the other samples contains at least onemolecule to be tested for binding. The thermal denaturation curve forthe receptor in the control well is the curve against which curves forall of the other experiments are compared.

The rate of screening is accelerated when the sample contains more thanone molecule to be tested for binding. For example, the screening rateis increased 20-fold when the sample contains a pool of 20 molecules.Samples which contain a binding molecule must then be divided intosamples containing a smaller collection of molecules to be tested forbinding. These divided collections must then be assayed for binding tothe target molecule. These steps must be repeated until a singlemolecule responsible for the original thermal shift is obtained.

Receptor denaturation can be measured by light spectrophotometry. When aprotein in solution denatures in response to heating, the receptormolecules aggregate and the solution becomes turbid. Thermally inducedaggregation upon denaturation is the rule rather than the exception.Aggregation generally complicates calorimetric experiments. Aggregation,however, is an advantage when using a spectrophotometric technology,because changes in turbidity can be measured by monitoring the change inabsorbance of visible or ultraviolet light of a defined wavelength.

Denaturation of a nucleic acid can be monitored using lightspectrophotometry. The change in hyperchromicity, which is the increasein absorption of light by polynucleotide solutions due to a loss ofordered structure, is monitored as a function of increase intemperature. Changes in hyperchromicity is typically assayed using lightspectrophotometry.

In another embodiment, however, fluorescence spectrometry is used tomonitor thermal denaturation. The fluorescence methodology is moresensitive than the absorption methodology.

The use of intrinsic protein fluorescence and fluorescence probemolecules in fluorescence spectroscopy experiments is well known tothose skilled in the art. See, for example, Bashford, C. L. et al.,Spectrophotometry and Spectrofluorometry: A Practical Approach, pp.91-114, IRL Press Ltd. (1987); Bell, J. E., Spectroscopy inBiochemistry, Vol. I, pp. 155-194, CRC Press (1981); Brand, L. et al.,Ann. Rev. Biochem. 41:843 (1972).

If the target molecule or receptor to be studied is a nucleic acid,fluorescence spectrometry can be performed using an ethidium bromidedisplacement assay (Lee, M. et al., J. Med. Chem. 36:863-870 (1993)). Inthis approach, ligand binding displaces ethidium bromide and results ina decrease in the fluorescent emission from ethidium bromide. Analternative approach is to use fluorescence resonance emission transfer.In the latter approach, the transfer of fluorescent energy, from a donorfluorophore on one strand of an oligonucleotide to an acceptorfluorophore on the other strand, is monitored by measuring the emissionof the acceptor fluorophore. Denaturation prevents the transfer offluorescent energy.

The fluorescence resonance emission transfer methodology is well knownto those skilled in the art. Fore example, see Ozaki, H. et al., NucleicAcids Res. 20:5205-5214 (1992); Clegg, RIM. et al., Proc. Natl. Acad.Sci. U.S.A. 90:2994-2998 (1993); Clegg, R. M. et al., Biochemistry31:4846-4856 (1993).

The element upon which the sample carrier is heated can be any elementcapable of heating samples rapidly and in a reproducible fashion. In thepresent invention, a plurality of samples is heated simultaneously. Theplurality of samples can be heated on a single heating elementAlternatively, the plurality of samples can be heated to a giventemperature on one heating element, and then moved to another heatingelement for heating to another temperature. Heating can be accomplishedin regular or irregular intervals. To generate a smooth denaturationcurve, the samples should be heated evenly, in intervals of 1 or 2° C.The temperature range across which the samples can be heated is from 25to 110° C. Spectral readings are taken after each heating step. Samplescan be heated and read by the spectral device in a continuous fashion.Alternatively, after each heating step, the samples may be cooled to alower temperature prior to taking the spectral readings. Preferably, thesamples are heated continuously and spectral readings are taken whilethe samples are being heated.

Spectral readings can be taken on all of the samples in the carriersimultaneously. Alternatively, readings can be taken on samples ingroups of at least two at a time. Finally, the readings can be taken onesample at a time.

In one embodiment, thermal denaturation is monitored by fluorescencespectrometry using an assay apparatus such as the one shown in FIG. 29.The instrument consists of a scanner and a control software system. Thesystem is capable of quantifying soluble and cell-associatedfluorescence emission. Fluorescence emission is detected by aphotomultiplier tube in a light-proof detection chamber. The softwareruns on a personal computer and the action of the scanner is controlledthrough the software. A precision X-Y mechanism scans the microplatewith a sensitive fiber-optic probe to quantify the fluorescence in eachwell. The microplate and samples can remain stationary during thescanning of each row of the samples, and the fiber-optic probe is thenis moved to the next row. Alternatively, the microplate and samples canbe moved to position a new row of samples under the fiber-optic probe.The scanning system is capable of scanning 96 samples in under oneminute. The scanner is capable of holding a plurality of excitationfilters and a plurality of emission filters to measure the most commonfluorophores. Thus, fluorescence emission readings can be taken onesample at a time, or on a subset of samples simultaneously. An alternateembodiment of the assay apparatus is shown in FIG. 33. The assayapparatus of the present invention will be described in more detailbelow.

The present invention is also directed to a product of manufacture whichcomprises a carrier having a multiplicity of containers within it. Theproduct of manufacture can be used to screen a combinatorial library forlead compounds which bind to the receptor of interest The combinatoriallibrary can be screened using the method according to the presentinvention.

In the product of manufacture, each of the containers contains a uniformamount of a receptor of interest In addition, each of these containerscontains a different compound from a combinatorial library at aconcentration which is at least 2-fold above the concentration of thereceptor. Preferably, the product of manufacture is a multiwellmicroplate or a multiplicity of multiwell microplates. If the receptoris a protein, each container may further contain a fluorescence probemolecule. If the receptor is a nucleic acid, each container may furthercontain ethidium bromide. Alternatively, the nucleic acid may be labeledwith a fluorophore.

Prior to use, the product of manufacture can be stored in any mannernecessary to maintain the integrity of the receptor of interest Forexample, the product of manufacture can be stored at a temperaturebetween −90° C. and room temperature. The receptor and compound can bestored in lyophilized form, in liquid form, in powdered form, or can bestored in glycerol. The product of manufacture may be stored either inthe light or in the dark.

The heat conducting element or block upon which the sample carrier isheated can be any element capable of heating samples rapidly andreproducibly. The plurality of samples can be heated on a single heatingelement. Alternatively, the plurality of samples can be heated to agiven temperature on one heating element, and then moved to anotherheating element for heating to another temperature. Heating can beaccomplished in regular or irregular intervals. To generate a smoothdenaturation curve, the samples should be heated evenly, in intervals of1 or 2° C. The temperature range across which the samples can be heatedis from 25 to 110° C.

In the present invention, a plurality of samples is heatedsimultaneously. If samples are heated in discrete temperature intervals,in a stairstep fashion, spectral readings are taken after each heatingstep. Alternatively, after each heating step, the samples may be cooledto a lower temperature prior to taking the spectral readings.Alternatively, samples can be heated in a continuous fashion andspectral readings are taken during heating.

Spectral readings can be taken on all of the samples on a carriersimultaneously. Alternatively, readings can be taken on samples ingroups of at least two at a time.

The present invention also provides an improved method for generatinglead compounds. After a compound or a combinatorial library of compoundshas been screened using the thermal shift assay, compounds which bind tothe target receptor are chemically modified to generate a second libraryof compounds. This second library is then screened using the thermalshift assay. This process of screening and generating a new librarycontinues until compounds that bind to the target receptor withaffinities in the K_(d) range of from 10⁻⁴ to 10⁻¹⁵ M are obtained.

A fluorescence emission imaging system can be used to monitor thethermal denaturation of a target molecule or a receptor. Fluorescenceemission imaging systems are well known to those skilled in the art. Forexample, the AlphaImager™ Gel Documentation and Analysis System (AlphaInnotech, San Leandro, Calif.) employs a high performancd charge coupleddevice camera with 768×494 pixel resolution. The charge coupled devicecamera is interfaced with a computer and images are anlayzed with Imageanalysis software. The ChemiImager™ (Alpha Innotech) is a cooled chargecoupled device that performs all of the functions of the AlphaImager™and in addition captures images of chemiluminescent samples and otherlow intensity samples. The ChemiImager™ charge coupled device includes aPentium processor (1.2 Gb hard drive, 16 Mb RAM), AlphaEase™ analysissoftware, a light tight cabinet, and a UV and white lighttrans-illuminator. For example, the MRC-1024 UV/Visible Laser ConfocalImaging System (BioRad, Richmond, Calif.) facilitates the simultaneousimaging of more than one fluorophore across a wide range of illuminationwavelengths (350 to 700 nm). The Gel Doc 1000 Fluorescent GelDocumentation System (BioRad, Richmond, Calif.) can clearly displaysample areas as large as 20×20 cm, or as small as 5×4 cm. At least two96 well microplates can fit into a 20×20 cm area. The Gel Doc 1000system also facilitates the performance of time-based experiments.

A fluorescence thermal imaging system can be used to monitor receptorunfolding in a microplate thermal shift assay. In this embodiment, aplurality of samples is heated simultaneously between 25 to 110° C. Afluorescence emission reading is taken for each of the plurality ofsamples simultaneously. For example, the fluorescence emission in eachwell of a 96 or a 384 well microplate can be monitored simultaneously.Alternatively, fluorescence emission readings can be taken continuouslyand simultaneously for each sample. At lower temperatures, all samplesdisplay a low level of fluorescence emission. As the temperature isincreased, the fluorescence in each sample increases. Wells whichcontain ligands which bind to the target molecule with high affinityshift the thermal denaturation curve to higher temperatures. As aresult, wells which contain ligands which bind to the target moleculewith high affinity fluoresce less, at a given temperature above theT_(m) of the target molecule in the absence of any ligands, than wellswhich do not contain high-affinity ligands. If the samples are heated inincremental steps, the flourescence of all of the plurality of samplesis simultaneously imaged at each heating step. If the samples are heatedcontinuously, the fluorescent emission of all of the plurality ofsamples is simultaneously imaged during heating.

A thermal shift assay can be performed in a volume of 100 μL volumes.For the following reasons, however, it is preferable to perform athermal shift assay in a volume of 10 μL. First, approximately 10-foldless protein is required for the miniaturized assay. Thus, only ˜5 to 40pmole of protein are required (0.1 μg to 1.0 μg for a 25 kDa protein)for the assay (i.e. 5 to 10 μL working volume with a target moleculeconcentration of about 1 to about 4 μM). Thus, 1 mg of protein can beused to conduct 1,000 to 10,000 assays in the miniaturized format. Thisis particularly advantageous when the target molecule is available inminute quantities.

Second, approximately 10-fold less ligand is required for theminiaturized assay. This advantage is very important to researchers whenscreening valuable combinatorial libraries for which library compoundsare synthesized in minute quantities. In the case of human α-thrombin,the ideal ligand concentration is about 50 μM, which translates into 250pmoles of ligand, or 100 ng (assuming a MW of 500 Da) of ligand perassay in the miniaturized format.

Third, the smaller working volume allows the potential of using largerarrays of assays because the miniaturized assay can fit into a muchsmaller area. For example, a 384 well (16×24 array) or 864 well (24×36array) plates have roughly the same dimensions as the 96 well plates(about 8.5×12.5 cm). The 384 well plate and the 864 well plate allowsthe user to perform 4 and 9 times as many assays, respectively, as canbe performed using a 96 well plate. Alternatively, 1536 well plates(32×48 arrays; Matrix Technologies Corp.) can be used. A 1536 well platewill facilitate sixteen times the throughput afforded by a 96 wellplate.

Thus, using the 1536 well plate configuration, the assay speed can beincreased by about 16 times, relative to the speed at which the assaycan be performed using the 96 well format. The 8×12 assay arrayarrangement (in a 96-well plate) facilitates the performance of 96assays/hr, or about 2300 assays/24 hours. The 32×48 array assayarrangement facilitates the performance of about 1536 assays hr., orabout 37,000 assays/24 hours can be performed using a 32×48 assay arrayconfiguration.

The assay volume can be 1-100 μL. Preferably, the assay volume is 1-50μL. More preferably, the assay volume is 1-25 μL. More preferably still,the assay volume is 1-10 μL. More preferably still, the assay volume is1-5 μL. More preferably still, the assay volume is 5 μL. Mostpreferably, the assay volume is 1 μL or 2 μL.

Preferably, the assay is performed in V-bottom polycarbonate plates orpolycarbonate dimple plates. A dimple plate is a plate that contains aplurality of round-bottom wells that hold a total volume of 15 μL.

One alternative to taking spectral readings over a temperature rangearound the T_(m) of the therapeutic target to obtain a full thermalunfolding curve for the ligand/target complex, in order to identifyshifts in T_(m) is to perform the assay at a single temperature near theT_(m) of the target molecule. In this embodiment, samples that emit lessfluorescence, relative to a control sample (containing a targetmolecule, but no candidate ligand) indicate that the candidate ligandbinds to the target molecule.

In this embodiment, the magnitude of a physical change associated withthe thermal denaturation of a target molecule resulting from heating isdetermined by generating a thermal denaturation curve for the targetmolecule as a function of temperature over a range of one or morediscrete or fixed temperatures. The physical change associated withthermal denaturation, for example, fluorescence emission, is measured.The magnitude of the physical change at the discrete or fixedtemperature for the target molecule in the absence of any ligand isnoted. The magnitude of the physical change in the presence of each of amultiplicity of different molecules, for example, combinatorialcompounds, is measured. The magnitude of the physical change associatedwith thermal denaturation of the target molecule in the presence of eachof the multiplicity of molecules is compared to magnitude of thephysical change obtained for the target molecule at the discrete orfixed temperature in the absence of any of the multiplicity of differentmolecules. The affinities of the multiplicity of different molecules areranked according to the change in the magnitude of the physical change.

The discrete or fixed temperature at which the physical change ismeasure can be any temperature that is useful for discriminating shiftsin thermal stability. Preferably, the discrete or fixed temperature isthe midpoint temperature T_(m) for the thermal denaturation curve forthe target molecule in the absence of any of the multiplicity ofdifferent molecules tested for binding to the target molecule.

The single temperature configuration is particularly advantageous if oneis interested in assaying a series of relatively high affinity ligands,which are the preferred compounds for candidates in clinical testing. Incases where a less stringent requirement for binding affinity ispreferred, however, one may increase the ligand concentration to 500 μMin order to identify ligands with K_(d)'s of 2.5 μM or higher affinity.

The single temperature embodiment offers a number of advantages. First,assay speed is increased by a factor often fold. Thus, as the 96 wellplate (8×12 array) assay facilitates about 96 assays per hour, thesingle temperature variation will facilitate about 1000 assays per hr.Using a 1536 well plate (32×48 array), as long as sample aliquoting canbe effected at the same rate for the 32×48 array system as in the 8×12array system, about 15,000 assays can be performed per hour.

Another alternative method for detecting the thermal unfoldingtransitions for the microplate thermal shift assay is through theintrinsic tryptophan (Trp) fluorescence of the target protein. Mostfluorescence emission plate readers, such as the CytoFluor II, usetungsten-halogen lamps as their light source. These lamps do not giveoff enough light near 280 nm to allow excitation of the intrinsic Trpresidues of proteins which absorb maximally near 280 nm. However, theBiolumin 960 (Molecular Dynamics) uses a Xenon-Arc lamp. The Xenon-Arclamp affords excitation at 280 nm and the measurement of emission at 350nm.

The methods and assay apparatus of the present invention are not limitedto assaying ligand-protein interactions. The methods and the assayapparatus can be used to rapidly assay any multi-variable system relatedto protein stabilization. For example, the methods and the assayapparatus of the present invention can be used to simultaneously assaythe binding of more than one compound or ligand to a target molecule.Using this approach, the additive effect of multiple-ligand binding canbe assessed. Positive and negative cooperativity can be determined. Toaccomplish this method, thermal shift assays are performed for a targetmolecule, such as a protein, in the absence of any ligands, in thepresence of a single ligand, and in the presence of two or more ligands.A thermal denaturation curve is generated for the protein alone and foreach combination of protein and ligand(s). The midpoint temperatureT_(m) is then determined for each curve. Each T_(m) is then compared toeach of the other T_(m)'s for the other curves. Alternatively, eachentire thermal denaturation curve is compared to each of the otherthermal denaturation curves. In either of these manners, the additivecontribution of more than one ligand to binding interaction or toprotein stability can be determined.

In a similar fashion, the additive contributions of one or morebiochemical conditions to protein stability can be determined. Thus, thepresent invention can be used to rapidly identify biochemical conditionsthat optimize protein stability, and hence shelf-life. of a protein.

Further, the methods and the assay apparatus of the present inventioncan be used to rank the efficacies of various biochemical conditions forrefolding or renaturing an unfolded or denatured protein. Thisembodiment addresses the need in the art for a reliable method forscreening for effective refolding or renaturing conditions.

For example, expression of recombinant DNA in a bacterial cell usuallyresults in the sequestration of recombinant protein into bacterialinclusion bodies (Marston, F. A. O., Biochem. J. 240:1-12 (1986)).Although other expression systems can be used instead of bacterialexpression systems, expression in bacterial cells remains the method ofchoice for the high-level production of recombinant proteins (Rudolph,R., Protein Engineering: Principles and Practices, pp. 283-298, JohnWiley & Sons (1995)). In many cases, recovery of recombinant proteinrequires that protein be isolated from inclusion bodies. Proteinpurification from inclusion bodies process necessitates the denaturationof recombinant protein. As a result, recombinant protein must berenatured or refolded under conditions suitable to generate the proteinin its native, fully functional form.

In each of these cases, denatured protein must be renatured or refoldedin order to be useful for further study or use. Unfortunately, onecannot easily predict the exact conditions under which a given proteinor fragment of the protein should be renatured. Each protein isdifferent. One must always resort to testing a number of differentcombinations of renaturing conditions before one can know which set ofconditions is optimal. Thus, it is desirable to have a reliable andrapid method for ranking the efficacies of various renaturingconditions.

Recombinant DNA technology has allowed the biosynthesis of a widevariety of heterologous polypeptides of interest in relatively largequantities through the recruitment of the bacterial protein expressionapparatus. However, the promise of cheap and abundant supplies ofcorrectly folded rare human proteins of high therapeutic value expressedin E. coli has foundered due to the overwhelmingly predominantaggregation of unfolded or partially unfolded target proteins intoinsoluble protein inclusion bodies. For recent reviews, see Rudolph, R.,& Lilie, H., FASEB J 10:49-56 (1995); Sadana, A., Biotechnology&Bioengineering 48:481-489 (1995); Jaenicke, R., Phil. Trans. Royal Soc.London Ser. B-Biol Sci 348:97-105 (1995)). Reasons for the prevailingself aggregation reaction in E. coli have centered on the relativelyhigh concentration of the heterologous protein (as high as 30% of theweight of the cell) found to various degrees in partially unfoldedstates. Thus, at the elevated protein concentrations of anoverexpressing E. coli strain, the exposed hydrophobic residues ofunfolded proteins are more likely to encounter other molecules withsimilarly exposed groups (inter-molecular reaction) than they are tosample self collapsed polypeptide conformations where these hydrophobicresidues are packed in a proper orientation (intra-molecular transitionstate) for proceeding to the fully folded native state (see FIG. 26).From this perspective, the insoluble protein inclusion bodies are seenas kinetically trapped side reaction products that thwart the preferredprotein folding process.

Techniques for isolating inclusion bodies, purifying recombinant proteinfrom inclusion bodies, and techniques for refolding or renaturingprotein are well known to those skilled in the art. For example, seeSambrook, J. et al., Molecular Cloning: a Laboratory Manual, pp.17.37-17.41, Cold Spring Harbor Laboratory Press (1989); Rudolph, R. etal., FASEB J. 10:49-56 (1995).

Another impediment to producing large quantities of correctly foldedproteins in E. coli is that the reducing redox potential of the E. colicytosol impedes the formation of disulfide bonds in vivo. The formationof disulfide bonds is an important co- and post-translational event inthe biosynthesis of many extracellular proteins that is often coupled toprotein folding. In addition, the cistrans proline isomerizationreaction has been demonstrated to be a rate determining step for correctfolding of certain proteins (Lin, L.-N., & Brandts, J. F., Biochemistry22:564-573 (1983)). As a result, partially folded intermediatesaccumulate in sufficient quantity in vivo that they aggregate andprecipitate into protein masses.

Cells employ a class of host proteins called molecular chaperonins thatassist in vivo protein folding by apparently preventing many of theunproductive side reactions discussed above with regard to inclusionbody formation, i.e. aggregation and improper disulfide bond formation.However, the E. coli chaperonin machinery, which is comprised in part bythe proteins, GroEL and GroES, presumably becomes overwhelmed by massiveoverexpression. Despite many attempts to correct this chaperonin deficitby coexpression of molecular chaperonins with the protein of interest(Rudolph, R., & Lilie, H., The FASEB J. 10:49-56 (1995)) positiveresults have been reported in only one case (Goloubinoff, P., et al.,Nature 342:884-889 (1989)).

Two hypotheses have been promoted to explain how GroEL and GroES assistin vivo protein folding. Under the first hypothesis, the Anfinsen cagehypothesis, the function of a molecular chaperonin is to provide aprotected environment where folding of the protein to its native statecan proceed without interference by pro-aggregation conditions in thecell (Martin, et al, Nature 352:36-42 (1991); Ellis, R. J., CurrentBiology 4:633-635 (1994)). Under the second hypothesis, the “iterativeannealing” hypothesis, the function of the chaperonin is to partlyunfold misfolded proteins (that is, kinetically trapped intermediates)with some of the energy of ATP hydrolysis being channeled into theconformational energy of the substrate polypeptide, forcing thepolypeptide into a higher energy state from which it could once againattempt to refold correctly after being released into solution (Todd, M.J. et al, Science 265:659-666 (1994); Jackson, et al, Biochemistry32:2554-2563 (1993); Weissman, J. S., et al., Cell 78:693-702 (1994);Weissman, J. S., & Kim, P.S., Science 253:1386-1393 (1991)).

The in vivo results discussed above are in many ways consistent with themore recent experiences with in vitro refolding of recombinantheterologous proteins expressed in E. coli. That is, while the primaryamino acid sequence of a protein may contain sufficient information todetermine its native folded conformation (Anfinsen, C. B., Science181:223-230 (1973)), the biochemical conditions in which the foldingreaction takes place can strongly influence the partitioning betweenunfolded, aggregated, and correctly folded forms.

For example, pH can be understood to influence the folding reaction byits effect on the long range electrostatic interactions summed in thefourth term of the equation (4). $\begin{matrix}{{\Delta \quad G_{fold}} = {{\Delta \quad G_{conf}} + {{\Sigma\Delta}\quad g_{i,{int}}{\Sigma\Delta}\quad g_{i,s}} + {\Delta \quad W_{el}} + \left( {\Delta \quad G_{bind}} \right)}} & \left( {{equation}\quad 4} \right)\end{matrix}$

where

ΔG_(conf)=conformational free energy (order/disorder term);

Δg_(i,int)=short range interactions (H-bonds, van der Wallsinteractions, salt bridges, cofactor binding, etc.);

Δg_(i,s)=short range interactions with solvent (hydrophobic effect,hydration of ions, etc.); and

ΔW_(el)=long range electrostatic interactions.

ΔG_(bind)=ligand binding free energy

As the pH of a protein solution is lowered below the pI for the protein,functional groups on the polypeptide become increasingly protonated, tothe point where the electrostatic repulsion between functional groupseventually out balances the other terms in the free energy equation(equation (4)), and the protein is no longer able to adopt the nativeconformation.

Another important biochemical parameter for protein folding is thesolvent, water, which repels aliphatic and aromatic side chains (andpossibly the main chain to some extent) to minimize their exposedsurface area. The influence of solvent over the folding reaction issummed in the third term of the free energy equation (equation (4)).Certain salts are known to increase the hydrophobic interaction amongprotein side chains in water solutions. The effect depends upon thenature of the ions following the Hofmeister series: Cations:Mg²⁺>Li⁺>Na+>K⁺>NH₄ ⁺. Anions: SO₄ ²⁻>HPO₄ ²⁻>acetate>citrate>tartrate>Cl⁻>NO₃−>ClO₃ ⁻>I⁻>ClO₄ ⁻>SCN⁻. Stabilizing Hofmeister anions,such as SO₄ ²⁻and HPO₄ ²⁻at 0.4 M have been found to increase the yieldof correctly folded proteins (Creighton, T. E., In: Proteins: Structuresand Molecular Properties, Freeman, New York, (1984)). This favorableoutcome for the native conformation of the protein has been attributedto the cations' and anions' “salting out” effect which leads to thepreferential hydration of the protein (Creighton, T. E., In: Proteins:Structures and Molecular Properties, Freeman, New York, (1984)).

Glycerol alters the solvation properties of water to favor the nativeconformation of proteins. The mechanism by which this occurs is theco-solvent exclusion and preferential hydration of the protein, notunlike the effect of salts of the salts of the Hofmeister series(Timasheff & Arakawa, In: Protein Structure, A Practical Approach, T. E.Creighton, ed., IRL Press Oxford, UK (1989), pp. 331-354).

Another example of how the environment influences protein folding is theeffect that known ligands and cofactors have on the yield of foldedprotein. Ligand binding has the effect of shifting the equilibrium froman unfolded state to a native-ligand complex through a coupling of thebinding free energy to that of the folding reaction. The role of metalions in the refolding of bovine carbonic anhydrase II has been described(Bergenhem & Carlsson, Biochim. Biopys. Acta 998:277-285 (1989)). Otherbiochemical parameters that have been shown to affect protein foldingare: protein concentration, temperature, glutathione redox buffers (GSH,GSSG), the presence of detergents, and the presence of other additives,such as glycerol, arginine-HCl, polyethylene glycol (PEG), and organicsolvents.

During incubation under refolding conditions, recombinant proteins canbe immobilized to solid phase support. This configuration resembles the“Anfinsen cage” hypothesis for the function of GroEL and GroES where anunfolded protein becomes temporarily immobilized in a protectedenvironment where folding to the native state can proceed withoutinterference from competing aggregation reactions. Confirmation ofprotein folding on solid supports has now come from two recent reportsin the literature. A poly-histidine tagged TIMP-2 protein could berefolded by dialysis while still bound to a metal chelate column (Negro,A. et al., FEBS Lett. 360:52-56 (1995)). A polyionic fusion peptideattached to the amino or carboxyl terminus of α-glucosidase allowedfolding while bound to heparin-Sepharose resin at about 5 mg/mL (Rudolph& Lilie, FASEB J. 10:49-56 (1995)). A polyionic arginine tagmetholdology for immobilicing and refolding α-glucosidase is disclosedin Stempfer, G. et al., Nature Biotechnology 14:329-334 (1996).

In the present invention, the thermal shift assay is used to rank theefficacy of various refolding or renaturng conditions. Each of amultiplicity of aliquots of a protein of interest, which has beenincubated under a variety of different biochemical folding conditions,are placed in a container in a multicontainer carrier. An aliquot of thenative, fully functional protein of known concentration is placed in thecontrol container. The samples can be placed in any multicontainercarrier. Preferably, each sample can be placed in a well of a multiwellmicroplate.

In considering the many biochemical variables that can influence theoutcome of the protein folding reaction, optimization of protein foldingis a multi-variable optimization problem, not unlike proteincrystallization and quantitative structure activity relationships (QSAR)in drug discovery. Multi-variable optimization problems require largenumbers of parallel experiments to collect as much data as possible inorder to influence a favorable response. In this regard, both proteincrystallization and QSAR analyses have greatly benefited from massscreening protocols that employ matrix arrays of incremental changes inbiochemical or chemical composition.

The present invention can be used to rank the efficacies of refolding orrenaturing conditions. Such conditions include, but are not limited to,the concentration of glycerol, the concentration of protein, the use ofagents which catalyze the formation of disulfide bond formation,temperature, pH, ionic strength, type of solvent, the use of thiols suchas reduced glutathione (GSH) and oxidized glutathione (GSSG), chaotropessuch as urea, guanidinium chlorides, alkyl-urea, organic solvents suchas carbonic acid amides, L-arginine HCl, Tris buffer, polyethyleneglycol, nonionic detergents, ionic detergents, zwitterionic detergents,mixed micelles, and a detergent in combination with cyclodextrin. Thepresent invention can be used regardless of whether a denaturation agentis removed from the protein using dialysis, column chromatographictechniques, or suction filtration.

Using a spectral thermal shift assay, the conditions which facilitateoptimal protein refolding can be determined rapidly. In this embodiment,the renatured protein samples and a control protein sample (i.e., asample of native protein in its fully functional form) are heated over atemperature range. At discrete temperature intervals, a spectral readingis taken. Alternatively, spectral readings can be taken during acontinuous, pre-determined temperature profile. A thermal denaturationcurve is constructed for each sample. The T_(m) for the thermaldenaturation curve of the native, fully functional protein isdetermined. The relative efficacies of the refolding conditions areranked according to the magnitude of the physical change associated withunfolding at the T_(m) of the native, fully functional protein, relativeto the magnitude of the physical change of a known quantity of thenative, fully functional protein at that T_(m). The magnitude ofphysical change used to measure the extent of unfolding (reflected onthe ordinate, or y-axis, of a thermal denaturation curve) corresponds tothe amount of correctly folded protein.

The present invention provides a method for screening biochemicalconditions that facilitate and optimize protein folding. To screenconditions for a given protein, it is first necessary to determine thethermal unfolding profile for a protein of interest. This isaccomplished by generating a denaturation curve using the microplatethermal shift assay. Various conditions can be opting, including pHoptimum, ionic strength dependence, concentration of salts of theHofmeister series, glycerol concentration, sucrose concentration,arginine concentration, dithiothreitol concentration, metal ionconcentration, etc.

Using the microplate thermal shift assay, one can determine one or morebiochemical conditions have an additive effect on protein stability.Once a set of biochemical conditions that facilitate an increase inprotein stability have been identified using the thermal shift assay,the same set of conditions can be used in protein folding experimentswith recombinant protein. See FIG. 27. If the conditions that promoteprotein stability in the thermal shift assay correlate with conditionsthat promote folding of recombinant protein, conditions can be furtheroptimized by performing additional thermal shift assays until acombination of stabilizing conditions that result in further increaseprotein stability are identified. Recombinant protein is then foldedunder those conditions. This process is repeated until optimal foldingconditions are identified. Protein stability is expected to correlatewith improved yields of protein folding. Yield of correctly foldedprotein can be determined using any suitable technique. For example,yield of correctly folded protein can be calculated by passing refoldedprotein over an affinity column, for example, a column to which a ligandof the protein is attached, and quantifying the amount of protein thatis present in the sample. In this way, folding conditions can beassessed for their additive contributions to correct folding. Thetransition state for the protein folding reaction resembles the nativeform of the protein more than the denatured form. This has beendemonstrated to be the case for may proteins (Fersht, A. R., Curr. Op.Struct. Biol. 7:3-9 (1997)).

The methods and the apparatus of the present invention provide a rapid,high throughput approach to screening for combinations of biochemicalconditions that favor the protein folding. The method does not requirecumbersome and time consuming steps that conventional approaches toprotein folding require. For example, using the method of the presentinvention, it is not necessary to dilute protein to large volumes andlow protein concentrations (˜10 μg/mL) in order to avoid aggregationproblems associated with conventional methods of recombinant proteinrefolding. Suppression of protein aggregation will allow for screeningbiochemical parameters that shift the protein folding equilibrium(between the unfolded and the folded forms of proteins) to the correctnative conformation.

Like protein stabilization, protein folding, ligand selection, and drugdesign, selection of conditions that promote protein crystallization isanother multi-variable optimization problem that is solved using themethods and the apparatus of the present invention.

The methods and the assay apparatus of the present invention are alsouseful for determining conditions that facilitate proteincrystallization. The crystallization of molecules from solution is areversible equilibrium process, and the kinetic and thermodynamicparameters are a function of the chemical and physical properties of thesolvent system and solute of interest (McPherson, A., In:Preparation andAnalysis of Protein Crystals, Wiley Interscience (1982); Weber, P. C.,Adv. Protein Chem. 41:1-36 (1991)) 1991). Under supersaturatingconditions, the system is driven toward equilibrium where the solute ispartitioned between the soluble and solid phase instead of the unfoldedand native states. The molecules in the crystalline phase pack inordered and periodic three dimensional arrays that are energeticallydominated by many of the same types of cohesive forces that areimportant for protein folding, i.e. van der Waals interactions,electrostatic interactions, hydrogen bonds, and covalent bonds (Moore,W. J., In: Physical Chemistry, 4th Ed., Prentice Hall, (1972), pp.865-898).

Thus, in many ways protein crystallization can be viewed as a higherlevel variation of protein folding where whole molecules are packed tomaximize cohesive energies instead of individual amino acid residues.Moreover, for both protein crystallization and protein folding, thecomposition of the solvent can make very important contributions to theextent of partitioning between the soluble (unfolded) and crystalline(native) forms. The cohesive interactions present in proteinmacromolecules and the role played by solvent in modulating theseinteractions for both protein folding and protein crystallization arecomplex and not fully understood at the present time. In this regard,biochemical conditions that promote protein stability and proteinfolding also promote protein crystallization.

For example, biochemical conditions that were found to increase thestability of D(II) FGF receptor 1 (FIGS. 19-24) correlate with theconditions that facilitated the crystallization of x-ray diffractionquality protein crystals. Conditions that were employed to obtaincrystals of D(II) FGFR1 protein (Lewankowski, Myslik, Bone, R. Springer,B. A. and Pantoliano, M. W., unpublished results (1997)) are shown inTable 1. Protein crystals were obtained in the pH range 7.4 to 9.2 inthe presence of the Hofmeister salt Li₂SO₄ (65 to 72%). Thesecrystallization conditions correlated with the pH optimum of about 8.0in FIG. 23. Other salts of the Hofmeister series such as Na₂SO₄,(NH₄)₂SO₄ and Mg₂SO₄ were also found useful as additives for loweringthe amount of Li₂SO₄ required as the precipitant Clearly, theseconditions for successful D(II) FGFR1 crystallization correlate closelywith the stabilizing conditions that were identified using themicroplate thermal shift assay.

Conditions that were identified as facilitating human α-thrombinstabilization also facilitate human α-thrombin protein crystallization.FIGS. 17A-D and 18 show the results of microplate thermal shift-assaysof conditions that facilitate human α-thrombin stability. Table 2contains a summary of the conditions identified by three differentinvestigators that facilitate crystallization of x-ray diffractionquality human α-thrombin crystals (Bode, W., et al, Protein Sci.1:426-471 (1992); Vijayalakshmi, J. et al., Protein Sci. 3:2254-22271(1994); and Zdanov, A. et al., Proteins: Struct. Funct. Genet.17:252-265 (1993)).

The conditions summarized in Table 2 correlate closely with theconditions identified in the microplate thermal shift assay asfacilitating human α-thrombin stability. Crystals formed near a pHoptimum of about 7.0. Furthermore, there is a clear preference for thepresence of 0.1 to 0.5 M NaCl (50% of the conditions) or 0.1 to 0.2 MNaHPO₄. This is consistent with the recently discovered Na⁺ binding site(Dang et al., Nature Biotechnology 15: 146-149 (1997)) and microplatethermal shift assay results in FIGS. 17A-D and 18. All of the humanα-thrombin samples described in Table 2 that have yielded good crystalsare complexed with a ligand, thereby further stabilizing the nativestructure of this protein beyond that acquired from the biochemicalconditions.

TABLE 1 D(II) FGFR1 Crystallization Conditions Buffer PrecipitantAdditive Protein Concentration 50 mM Hepes pH 7.4 72% Li2SO4 10 mg/ml(10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 72% Li2SO4 3.4 mM ZnSO4 10 mg/ml(10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 68% LI2SO4 1% PEG 8000 10 mg/ml(10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 66% Li2SO4 3.4 mM Na2SO4 10mg/ml (10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 66% Li2SO4 5.3 mM(NH4)2SO410 mg/ml (10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 66% Li2SO4 2.1 mM MgSO410 mg/ml (10 mM Hepes pH 7.5) 10 mM Tris Hcl, 65% Li2SO4 10 mg/ml (10 mMHepes pH 7.5) pH 8.0 20 mM glycine, 68% Li2SO4 10 mg/ml (10 mM Hepes pH7.5) pH 5.2

Protein crystallization is a slow and tedious process that hashistorically been the rate determining step for the x-ray diffractiondetermination of protein and nucleic acid structures. The method andapparatus of the present invention facilitate the rapid, high-throughputelucidation of conditions that promote the stability of a given proteinand thus the formation of X-ray quality protein crystals.

When a protein is more stable, it has fewer thermodynamic motions thatinhibit packing into a crystal lattice. With fewer motions, the proteinfits better into a crystal lattice. Using conventional crystallizationmethods, crystallization experiments are set up at room temperature forweeks at a time. Over time, protein unfolding occurs. Using the methodsof the present invention, conditions that stabilize a protein areexamined over a temperature range. Conditions that shift the thermalunfolding curve to higher temperature will lower extent of unfoldingthat occurs while the crystallization process occurs.

TABLE 2 Human α- Crystallization Conditions thrombin Protein ComplexBuffer Salt Precipitant Additive Conc. Comment Vijayalakshmi et al.MDL-28050 75 mM NaHPO4 pH 7.3 0.375 M NaCl 1 mM NaN3 3 mg/ml protein(2.2 Å) 100 mM NaHPO4 pH 7.3 24% PEG 4000 1 mM NaN3 well Hirugen/Hirulog1 50 mM NaHPO4 pH 7.3 0.375 M NaCl .5 mM NaN3 3-3.7 mg/ml protein (2.3Å) 0.1 M NaHO4 pH 7.3 28% PEG 8000 1 mM NaN3 well FPAM + Hirugen 0.1 MNaHPO4 pH 7.3 5 mg/ml protein (2.5 Å) 0.1 M NaHPO4 pH 7.3 28% PEG 8000well Hirulog 3 75 mM NaHPO4 pH 7.3 0.38 M NaCl 1 mM NaN3 5 mg/ml protein(2.3 Å) 0.1 M NaHPO4 pH 7.3 24% PEG 8000 1 mM NaN3 well Bode et al.NAPAP 0.1 M KHPO4 pH 8.0 10 mg/ml protein (2.3 Å) 0.1 M KHPO4 pH 8.0 1.9M NH4SO4 PPACK 2 mM MOPS pH7 0.1 M NaCl 0.5% NaN3 10 mg/ml protein (1.9Å) 0.2 M PO4 pH 6-7 0.5 M NaCl 20% PEG 6000 well Zdanov et al.Hirutonin-2 50 mM NaHPO4 pH 5.5 0.1 M NaCl 10 mg/ml Protein (2.1 Å) 0.1M Na Citrate pH 5.5 0.1 M NaCl 24% PEG 4000 well

Overview of Assay Apparatus

The assay apparatus of the present invention is directed to an automatedtemperature adjusting and spectral emission receiving system thatsimultaneously adjusts the temperature of a multiplicity of samples overa defined temperature range and receives spectral emission from thesamples. The assay apparatus of the present invention is particularlyuseful for performing microplate thermal shift assays of proteinstability. The assay apparatus of the present invention can be used topractice all of the methods of the present invention.

The assay apparatus of the present invention replaces separate heatingdevices and spectral emission receiving devices. In contrast to otherdevices, the assay apparatus of the present invention can be configuredto simultaneously adjust the temperature of a multiplicity of samplesand receive spectral emissions from the samples during adjustment oftemperature in accordance with a predetermined temperature profile.

After heat denaturation, reversibly folding proteins partially or fullyrefold after heat denaturation. Refolding precludes meaningfulmeasurements in a thermal shift assay. Using the assay apparatus of thepresent invention, however, one can assay reversibly folding proteins ina thermal shift assay. That is because in the assay apparatus of thepresent invention, spectral measurements are taken while the protein isbeing heated. And protein refolding does not occur.

In such a configuration, the assay apparatus of the present inventionincludes a sensor which is positioned over a movable heat conductingblock upon which an array of samples is placed. A relative movementmeans, such as a servo driven armature, is used to move the sensor sothat the sensor is sequentially positioned over each sample in the arrayof samples. The sensor receives spectral emissions from the samples.

The assay apparatus of the present invention can be configured so thatit contains a single heat conducting block. Alternatively, the assayapparatus can be configured so that it contains a plurality of heatconducting blocks upon a movable platform. The platform may be atranslatable platform that can be translated, for example, by a servodriven linear slide device. An exemplary linear slide device is model SAA5M400 (IAI America, Torrance, Calif.). In this embodiment, the sensorreceives spectral emissions from each of the samples on a given heatconducting block. The platform is then translated to place another heatconducting block and its accompanying samples under the sensor so thatit receives spectral emissions from each of the samples on that heatingblock. The platform is translated until spectral emissions are receivedfrom the samples on all heat conducting blocks.

Alternatively, the platform may by a rotatable platform that may berotated, for example, by a servo driven axle. In the latter embodiment,the sensor receives spectral emissions from each of the samples on agiven heat conducting block. The platform is then rotated to placeanother heat conducting block and its accompanying samples under thesensor so that it receives spectral emissions from each of the sampleson that heating block. The platform is rotated until spectral emissionsare received from the samples on all heat conducting blocks.

System Description

FIG. 29 shows a schematic diagram of one embodiment of an assayapparatus 2900 of the present invention. Assay apparatus 2900 includes aheat conducting block 2912 that includes a plurality of wells 2920 for aplurality of samples 2910. Heat conducting block 2912 is composed of amaterial that has a relatively high coefficient of thermal conductivity,such as aluminum, stainless steel, brass, teflon, and ceramic.

Thus, heat conducting block 2912 can be heated and cooled to a uniformtemperature but will not be thermally conductive enough to requireexcess heating or cooling to maintain a temperature.

Assay apparatus 2900 also includes a light source 2906 for emitting anexcitatory wavelength of light, shown generally at 2916, for thesamples. Light source 2906 excites samples 2910 with excitatory light2916. Any suitable light source can be used. For example atungsten-halogen lamp can be used. Alternatively, a Xenon-arc lamp, suchas the Biolumin 960 (Molecular Dynamics) can used.

Alternatively, a high pressure mercury (Hg) Lamp can be used. Highpressure mercury lamps emit light of higher intensity than Xenon (Xe)lamps. The intensity of light from a high pressure mercury lamp isconcentrated in specific lines, and are only useful if the Hg lines areat suitable wavelengths for excitation of particular fluorophores.

Some fluorescent plate readers employ lasers for excitation in thevisible region of the electromagnetic spectrum. For example, theFluorImager™ (Molecular Dynamics, Palo Alto, Calif.) is such a device.This technology is useful when using fluorescent dyes that absorb energyat around 480 nm and emit energy at around 590 nm. Such a dye could thenbe excited with the 488 nm illumination of standard argon, argon/kryptonlasers. For example,1,1-dicyano-2-[6di-methylamino)naphthalen-2-yl]propene (DDNP) is such adye. The advantage in using a laser is that a laser is characterized byvery high intensity light, which results in an improved signal to noiseratio.

Excitatory light 2916 causes a spectral emission 2918 from samples 2910.Spectral emission 2918 can be electromagnetic radiation of anywavelength in the electromagnetic spectrum. Preferably, spectralemission 2918 is fluorescent, ultraviolet, or visible light. Mostpreferably, spectral emission 2918 is fluorescence emission. Spectralemission 2918 is received by a photomultiplier tube 2904.Photomultiplier tube 2904 is communicatively and operatively coupled toa computer 2914 by an electrical connection 2902. Computer 2914functions as a data analysis means for analyzing spectral emission as afunction of temperature.

As discussed above, the spectral receiving means or sensor of the assayapparatus of the present invention can comprise a photomultiplier tube.Alternatively, the spectral receiving means or sensor can include acharge coupled device or a charge coupled device camera. In stillanother alternative, the spectral receiving means or sensor can includea diode array.

An alternate embodiment of the assay apparatus of the present inventionis shown in FIG. 30. In the embodiment shown in FIG. 30, a chargecoupled device (CCD) camera 3000 is used to detect spectral emission2918 from samples 2910. CCD camera 3000 can be any CCD camera suitablefor imaging fluorescent emissions. For example, suitable CCD cameras areavailable from Alpha-Innotech (San Leandro, Calif.), Stratagene (LaJolla, Calif.), and BioRad (Richmond, Calif.). For measuring fluorescentemission in the microplate thermal shift assay, one alternative to afluorescent plate reader is a charge coupled device (CCD). For example,high resolution CCD cameras can detect very small amounts ofelectromagnetic energy, whether it originates from distance stars, isdiffracted by crystals, or is emitted by fluorophores. A CCD is made ofsemi-conducting silicon. When photons of light fall on it, freeelectrons are released. As an electronic imaging device, a CCD camera isparticularly suitable for fluorescence emission imaging because it candetect very faint objects, affords sensitive detection over a broadspectrum range, affords low levels of electromagnetic noise, and detectssignals over a wide dynamic range—that is, a charge coupled device cansimultaneously detect bright objects and faint objects. Further, theoutput is linear so that the amount of electrons collected is directlyproportional to the number of photons received. This means that theimage brightness is a measure of the real brightness of the object, aproperty not afforded by, for example, photographic emulsions.

When a fluorescence imaging camera or a CCD camera is used, excitatorylight 2916 can be a suitable lamp that is positioned over the pluralityof samples 2910. Alternatively, excitatory light 2916 can be a suitablelamp that is positioned under the plurality of samples 2910. In anotheralternative embodiment, excitatory light 2916 can be delivered to eachsample 2910 by a plurality of fiber optic cables. Each fiber optic cableis disposed through one of a plurality of tunnels in conducting block2912. Thus, each of samples 2910 receives excitatory light 2916 througha fiber optic cable.

As shown in FIG. 30, source 2906 excites samples 2910 with excitatorylight 2916. Excitatory light 2916 causes spectral emission 2918 fromsamples 2910. Spectral emission 2918 is filtered through an emissionfilter 3002. Emission filter 3002 filters out wavelengths of spectralemission 2918 that are not to be monitored or received by CCD camera3000. CCD camera 3000 receives the filtered spectral emission 2918 fromall of samples 2910 simultaneously. For simplicity and ease ofunderstanding, only spectral emissions form one row of samples 2910 areshown in FIG. 30. CCD camera 3000 is communicatively and operativelycoupled to computer 2914 by electrical connection 2902.

With reference now to FIG. 31, one embodiment of assay apparatus 2900 isshown in more detail. As shown in FIG. 31, many apparatus components areattached to a base 3100. A heat conducting block relative movement means3128 is used to move heat conducting block 2912 in directions 3150 and3152. Heat conducting block relative movement means 3128 iscommunicatively and operatively connected to a servo controller 3144.Activation of heat conducting block relative movement means 3128 byservo controller 3144 moves heat conducting block 2912 in directions3150 and 3152. Servo controller 3144 is controlled by a computercontroller 3142. Alternatively, computer 2914 could be used to controlservo controller 3144.

A sensor is removably attached to a sensor armature 3120. An exemplarysensor is a fiber optic probe 3122. Fiber optic probe 3122 includes afiber optic cable capable of transmitting receiving excitatory light2916 to samples 2910, and a fiber optic cable capable of receivingspectral emission 2918 from samples 2910. Electromagnetic radiation istransmitted from excitatory light source 2906 to fiber optic probe 3122by excitatory light input fiber optic cable 3108. In one embodiment ofthe present invention, a spectral receiving means comprisingphotomultiplier tube 2904 is used to detect spectral emission fromsamples 2910. In this embodiment, electromagnetic radiation istransmitted from fiber optic probe 3122 to photomultiplier tube 2904 byfiber optic cable 3110. In an alternative embodiment of the presentinvention, CCD camera 3002 is used to detect spectral emission fromsamples 2910. In this embodiment, fiber optic cable 3110 is notrequired.

A temperature sensor 3124 is removably attached to sensor armature 3120.Temperature sensor 3124 is communicatively and operably linked to atemperature controller 3162. Temperature sensor 3124 monitors thetemperature of heat conducting block 2912 and feeds temperatureinformation back to temperature controller 3162. Temperature controller3162 is connected to heat conducting block 2912 by a thermoelectricconnection 3164. Under the action of temperature controller 3162, thetemperature of heat conducting block 2912 can be increased, decreased,or held constant. Particularly, the temperature of heat conducting block2912 can be changed by temperature controller 3162 in accordance with apre-determined temperature profile. Preferably, temperature computercontroller 3162 is implemented using a computer system such as thatdescribed below with respect to FIG. 37.

As used herein, the term “temperature profile” refers to a change intemperature over time. The term “temperature profile” encompassescontinuous upward or downward changes in temperature, both linear andnon-linear changes. The term also encompasses any stepwise temperaturechange protocols, including protocols characterized by incrementalincreases or decreases in temperature during which temperature increasesor decreases are interrupted by periods during which temperature ismaintained constant. In the apparatus of the present invention, thetemperature profile can be pre-determined by programing temperaturecomputer controller 3162. For example, temperature profiles can bestored in a memory device of temperature controller 3162, or input totemperature controller 3162 by an operator.

A sensor armature relative movement means 3130 is used to move sensorarmature 3120 in directions 3154 and 3156. A sensor armature servocontroller 3118 is fixedly connected to excitatory light filter housing3160. Activation of sensor armature servo controller 3118 moves fiberoptic probe 3122 in directions 3154 and 3156. It would be readilyapparent to one of ordinary skill in the relevant art how to configureservo controllers to move heat conducting block 2912 and sensor armature3120. It should be understood that the present invention is not limitedto the use of servo controllers for movement of heat conducting block2912 and sensor armature 3120, and other suitable means known to one ofskill in the art can also be used, such as a motor.

Servo controllers 3118 and 3144 are both communicatively and operativelyconnected to computer controller 3142. Computer controller 3142 controlsthe movement of sensor armature 3120 in directions 3154 and 3156. Inaddition, computer controller 3142 controls the movement of heatconducting block relative movement means 3128 in directions 3150 and3152.

In the assay apparatus of the present invention, excitatory light source2906 is used to excite samples 2910. Excitatory light source 2906 iscommunicatively and operably connected to excitatory light filter 3104,which is contained within excitatory light filter housing 3160.Excitatory light filter 3104 filters out all wavelengths of light fromexcitatory light source 2906 except for the wavelength(s) of light thatare desired to be delivered by fiber optic probe 3122 to samples 2910.An excitatory light filter servo controller 3106 controls the apertureof excitatory light filter 3104. Excitatory light source 2906 andexcitatory light filter servo controller 3106 are communicatively andoperatively connected to excitatory light computer controller 3102.Computer controller 3102 controls the wavelength of excitatory lighttransmitted to samples 2910 by controlling excitatory light filter servocontroller 3106. Excitatory light 2916 is transmitted through excitatorylight input fiber optic cable 3108 to fiber optic probe 3122 fortransmission to samples 2912.

Spectral emission 2918 from samples 2910 is received by fiber opticprobe 3122 and is transmitted to a spectral emission filter 3114 byoutput fiber optic cable 3110. Spectral emission filter 3114 iscontained within a spectral emission filter housing 3166. Spectralemission filter housing 3166 is disposed on photomultiplier tube housing3168. Photomultiplier tube housing 3168 contains photomultiplier tube2904. A spectral emission servo controller 3112 controls the aperture ofspectral emission filter 3114, thereby controlling the wavelength ofspectral emission 2918 that is transmitted to photomultiplier tube 2904.Spectral emission servo controller 3112 is controlled by a computercontroller 3170.

Spectral emission 2918 from samples 2910 is transmitted fromphotomultiplier tube 2904. Electrical output 3140 connectsphotomultiplier tube 2904 to electric connection 2902. Electricconnection 2902 connects electrical output 3140 to computer 2914. Drivenby suitable software, computer 2914 processes the spectral emissionsignal from samples 2910. Exemplary software is a graphical interfacethat automatically analyzes fluorescence data obtained from samples2910. Such software is well known to those of ordinary skill in the art.For example, the CytoFluor™ fluorescence multi-well plate reader(PerSeptive Biosystems, Framingham, Mass.) utilizes the Cytocalc™ DataAnalysis System (PerSeptive Biosystems, Framingham, Mass.). Othersuitable software includes, MicroSoft Excel or any comparable software.

FIGS. 32A-C illustrate one embodiment of a thermal electric stage orheat conducting block for the assay apparatus of the present invention.FIG. 32A shows a side view of heat conducting block 2912 and a heatconducting wire 3206. FIG. 32B shows a top view of heat conducting block2912 and heat conducting wire 3206. Heat conducting wire 3206 is atemperature adjusting element that adjusts the temperature of heatconducting block 2912. By means readily known to one of skill in theart, temperature controller 3162 causes heat conducting wire 3206 toincrease or decrease in temperature, thereby changing the temperature ofheat conducting block 2912. For example, an exemplary temperaturecontroller is a resistance device that converts electric energy intoheat energy. Alternatively, the heating element can be a circulatingwater system, such as that disclosed in U.S. Pat. No. 5,255,976, thecontent of which is incorporated herein by reference. In anotheralternative, the temperature adjusting element can be a heat conductingsurface upon which heat conducting block 2912 is disposed. Particularly,the temperature of heat conducting wire 3206 can be changed bytemperature controller 3162 in accordance with a pre-determinedtemperature profile. Temperature controller 3162 is preferablyimplemented using a computer system such as that described below withrespect to FIG. 37. Alternatively, computer 2914 could be used toimplement temperature controller 3162. An exemplary set ofspecifications for temperature controller 3162 and heat conducting block2912 is as follows:

resolution  0.1° C. accuracy +0.5° C. stability  0.1° C. repeatability 0.1° C.

Temperature controller 3162 changes temperature in accordance with atemperature profile as discussed below with respect to FIGS. 36A and36B.

The temperature of heat conducting block 2912 can be controlled suchthat a uniform temperature is maintained across the heat conductingblock. Alternatively, the temperature of heat conducting block 2921 canbe controlled such that a temperature gradient is established from oneend of the heat conducting block to the other. Such a technique isdisclosed in U.S. Pat. Nos. 5,255,976 and 5,525,300, the entirety ofboth of which is incorporated by reference.

Heat conducting block 2912 is preferably configured with plurality ofwells 2920 for samples 2910 to be assayed. In one embodiment, each ofwells 2920 is configured to receive a container containing one ofplurality of samples 2910. Alternatively, heat conducting block 2912 isconfigured to receive a container containing plurality of samples 2910.An exemplary container for containing plurality of samples 2910 is amicrotiter plate.

In yet a further alternate embodiment, heat conducting block 2912 isconfigured to receive a heat conducting adaptor that is configured toreceive a container containing one or more of samples 2910. The heatconducting adaptor is disposed on heat conducting block 2912, and thecontainer containing samples 2910 fits into the heat conducting adaptor.FIGS. 32C-E show three exemplary configurations of a heat conductingadaptor. An adaptor 3200 is configured with round-bottomed wells. Anadaptor 3202 is configured with square-bottom wells. An adaptor 3204 isconfigured with V-shaped wells. For example, adaptor 3200 can receive aplurality of round-bottom containers, each containing one sample.Similarly, adaptor 3202 can receive a plurality of square-bottomcontainers, and adaptor 3204 can contain a plurality of V-shaped bottomcontainers. Adaptors 3200, 3202, and 3204 can also receive a carrier fora multiplicity of round-bottom containers. An exemplary carrier is amicrotitre plate having a plurality of wells, each well containing asample. When heat conducting block 2912 is heated, heat conductingadaptors 3200, 3202, or 3204 are also heated. Thus, the samplescontained in the containers that fit within adaptors 3200, 3202, or 3204are also heated. Adaptors 3200, 3202, and 3204 can accept standardmicroplate geometries.

Another embodiment of the assay apparatus of the present invention isshown in FIG. 33. In this embodiment, a plurality of heat conductingblocks 2912 is mounted on a rotatable platform or carousel 3306.Alternatively, the platform can be a translatable platform. Platform orcarousel 3306 can be composed of a heat conducting material, such as thematerial that heat conducting block 2912 is composed of. Although sixheat conducting blocks are shown in FIG. 33, this number is exemplaryand it is to be understood that any number of heat conducting blocks canbe used. As shown in FIG. 33, an axle 3308 is rotatably connected tobase 3100. Rotatable platform 3306 is axially mounted to rotate aboutaxle 3308. Rotation of axle 3308 is controlled by a servo controller3312. Servo controller 3312 is controlled by a computer controller 3314in a manner well known to one of skill in the relevant arts. Computercontroller 3314 causes servo controller 3312 to rotate axle 3308 therebyrotating rotatable platform 3306. In this manner, heat conducting blocks2912 are sequentially placed under fiber optic probe 3122.

Each of the plurality of heat conducting blocks 2912 can be controlledindependently by temperature controller 3162. Thus, the temperature of afirst heat conducting block 2912 can be higher or lower than thetemperature of a second heat conducting block 2912. Similarly, thetemperature of a third heat conducting block 2912 can be higher or lowerthan the temperature of either first or second heat conducting block2912.

In a manner similar to that described above for FIG. 31, relativemovement means 3130 is also used to move sensor armature 3120 indirections 3150 and 3152 so that fiber optic probe 3122 can be moved todetect spectral emission from samples 2910. A second sensor armaturerelative movement means 3316 is used to move sensor armature 3120 indirections 3154 and 3156.

The temperature of heat conducting blocks 2912 is controlled bytemperature controller 3162. Temperature controller 3162 is connected torotatable platform 3306 by connection 3164 to heat conducting blocks2912. Under the action of temperature controller 3162, the temperatureof heat conducting blocks 2912 can be increased and decreased.Alternatively, temperature controller 3162 can be configured to adjustthe temperature of rotatable platform 3306. In such a configuration,when rotatable platform 3306 is heated, heat conducting blocks 2912 arealso heated. Alternatively, the temperature of each of heat conductingblocks 2912 can be controlled by a circulating water system such as thatnoted above.

In a manner similar to that illustrated in FIG. 31, excitatory lightsource 2906 is used to excite samples 2910. Excitatory light source 2906is communicatively and ably connected to excitatory light filter 3104,which is contained within excitatory light filter housing 3160.Excitatory light filter 3104 filters out all wavelengths of light fromexcitatory light source 2906 except for the wavelength(s) of light thatare desired to be delivered by fiber optic probe 3122 to samples 2910.An excitatory light filter servo controller 3106 controls the apertureof excitatory light filter 3104. Excitatory light source 2906 andexcitatory light filter servo controller 3106 are communicatively andoperatively connected to excitatory light computer controller 3102.Computer controller 3102 controls the wavelength of excitatory lighttransmitted to samples 2910 by controlling excitatory light filter servocontroller 3106. Excitatory light 2916 is transmitted through excitatorylight input fiber optic cable 3108 to fiber optic probe 3122 fortransmission to samples 2912.

Spectral emission 2918 from samples 2910 is received by fiber opticprobe 3122 and is transmitted to spectral emission filter 3114 by fiberoptic cable 3110. Spectral emission servo controller 3112 controlsspectral emission filter 3114 aperture and thus controls the wavelengthof spectral emission that is transmitted to photomultiplier tube 2904.In a manner similar to that explained for FIG. 31, spectral emissionservo controller 3112 is controlled by computer controller 3170.

The assay apparatus of the present invention can detect spectralemission from samples 2910 one sample at a time or simultaneously from asubset of samples 2910. As used herein, the term “subset of samples”refers to at least two of samples 2910. To detect spectral emissionsimultaneously from a subset of samples in an embodiment of the assayapparatus of the present invention comprising photomultiplier tube 2904,a plurality of excitatory light filters 3104, excitatory light inputfiber optic cables 3108, emission light output fiber optic cables 3110,and emission light filters 3114 must be used.

The spectral emission signal is transmitted from photomultiplier tube2904 to computer 2914. Photomultiplier tube 2904 is communicatively andoperatively coupled to computer 2914 by electrical connection 2902.Connection 2902 is connected to photomultiplier tube 2904 throughelectrical output 3140. Computer 2914 functions as a data analysis meansfor analyzing spectral emission as a function of temperature.

FIG. 34 illustrates a top view of the assay apparatus shown in FIG. 33with a housing 3400 that covers the apparatus. A door 3402 opens toreveal samples 2910. Door 3402 can be a hinge door that swings open.Alternatively, door 3402 can be a sliding door that slides open. A sideview of the assay apparatus shown in FIGS. 33 and 34 is illustrated inFIG. 35. Cover 3400 is disposed on top of base 3100. Cover 3400 can bemade of any suitable material. For example, cover 3400 can be made ofplexiglass, fiberglass, or metal.

FIGS. 36A and 36B illustrate a temperature profile and how thetemperature profile is implemented using the assay apparatus of thepresent invention. FIG. 36A illustrates a temperature profile 3600 thatshows the temperature of heat conducting blocks 2912 as a function oftime. Heat conducting blocks 2912 and samples 2910 are heated in acontinuous fashion in accordance with temperature profile 3600.Alternatively, rotatable platform 3306 can be heated along with heatconducting blocks 2912. Preferably, temperature profile 3600 is linear,with temperatures ranging from about 25° C. to about 110° C.

Alternatively, temperature profile 3600 can be characterized byincremental, stair step increases in temperature, in which heatconducting blocks 2912 and samples 2910 are heated to a predeterminedtemperature, maintained at that temperature for a predetermined periodof time, and than heated to a higher pre-determined temperature. Forexample, temperature can be increased from 0.5° C. to 20° C. per minute.Although the temperature range from about 25° C. to about 110° C. isdisclosed, it is to be understood that the temperature range with whicha given target molecule, for example, a protein, is to be heated togenerate a thermal denaturation curve can readily be determined by oneof ordinary skill in the art. The length of time over which temperatureprofile 3600 is accomplished will vary, depending on how many samplesare to be assayed and on how rapidly the sensor that receives spectralemission 2918 can detect spectral emission 2918 from samples 2910. Forexample, an experiment in which each of six heat conducting blocks 2912holds a total of 96 samples 2910, (for a total of 576 samples), and inwhich samples are scanned using a fluorescent reader device having asingle fiber optic probe, and in which the temperature profile is from38° C. and 76° C., would take approximately 38 minutes to perform usingthe apparatus shown in FIG. 33.

While heating in accordance with temperature profile 3600, spectralemission 2918 from each sample 2910 in a first heat conducting block2912 is received through fiber optic probe 3122. As illustrated in FIG.36B, after emissions from all of samples 2910 in first heat conductingblock 2912 have been received, platform 3306 is rotated to move the nextheat conducting block 2912 under fiber optic probe 3122 and spectralemission 2918 from samples 2910 is received by fiber optic probe 3122.This process is continued until reception of spectral emissions from allsamples in all heat conducting blocks 2912 is complete. Spectralemission from samples 2910 on each heat conducting block 2912 can bereceived one at a time, simultaneously from a subset of samples,simultaneously from one row of samples at a time, or all of the samplesat one time.

Computer Program Implementation of the Preferred Embodiments

The present invention may be implemented using hardware, software, or acombination thereof, and may be implemented in a computer system orother processing system. A flowchart 3800 for implementation of oneembodiment of the present invention is shown in FIG. 38. Flowchart 3800begins with a start step 3802. In a step 3804, temperature profile 3600is initiated. For example, temperature controller 3162 causes thetemperature of heat conducting block 2912 to increase. In a step 3806, asensor such as fiber optic probe 3122 or CCD camera 3000 is moved over asample 2910, row of samples 2910, or all of samples 2910. In a step3808, excitatory light is transmitted to sample(s) 2910 using excitatorylight source 2906. In a step 3810, spectral emission is received by thesensor from sample(s) 2910. In a decision step 3812, it is determinedwhether spectral emission 2918 has been received from all of thesamples, rows of samples, in one heat conducting block 2912. If spectralemission 2918 has not been received from all of the samples or rows ofsamples, the sensor is moved over the next sample or row of samples in astep 3814. Processing then continues at step 3808 to transmit excitatorylight 2916. Processing then continues to a step 3810 to receive spectralemission 2918 from sample(s) 2910.

If spectral emission 2918 has been received from all of samples or rowsof samples, processing continues to a decision step 3816. In decisionstep 3816, it is determined whether spectral emission 2918 has beenreceived from samples in all heat conducting blocks. If not, rotatableplatform 3306 is rotated in a step 3818 to place the next heatconducting block 2912 and samples 2910 contained therein under thesensor. Steps 3806 through 3818 are followed until spectral emission2918 has been received from all of the samples in all of heat conductingblocks 2912. Processing then continues to a step 3820, in whichtemperature profile 3600 is completed and processing ends at a step3822.

A flowchart 3900 for implementation of an alternate embodiment of thepresent invention is shown in FIG. 39. In this embodiment, a sensor forsimultaneously receiving spectral emission 2918 from all of samples 2910on heat conducting block 2912, such as CCD camera 3000, is positionedover heat conducting block 2912. Flowchart 3900 begins with a start step3902. In a step 3904, temperature profile 3600 is initiated. Forexample, temperature controller 3162 causes the temperature of heatconducting block 2912 to increase. In a step 3906, excitatory light istransmitted to sample(s) 2910 using excitatory light source 2906. In astep 3908, spectral emission is received by CCD camera 3000 fromsample(s) 2910. In a decision step 3910, it is determined whetherspectral emission 2918 has been received from all of heat conductingblocks 2912. If not, rotatable platform 3306 is rotated in a step 3912to place the next heat conducting block 2912 and samples 2910 containedtherein under CCD camera 3000. Steps 3906 through 3912 are followeduntil spectral emission 2918 has been received from samples 2910 in allof heat conducting blocks 2912. Processing then continues to a step3914. In step 3914, temperature profile 3600 is completed and processingends at a step 3916.

As stated above, the present invention may be implemented usinghardware, software, or a combination thereof, and may be implemented ina computer system or other processing system. An exemplary computersystem 3702 is shown in FIG. 37. Computer controllers 3102, 3142, 3162,3170, or 3314, can be implemented using one or more computer systemssuch as computer system 3702.

After reading this description, it will become apparent to a personskilled in the relevant art how to implement the invention using othercomputer systems and/or computer architectures. Computer system 3702includes one or more processors, such as processor 3704. Processor 3704is connected to a communication bus 3706.

Computer system 3702 also includes a main memory 3708, preferably randomaccess memory (RAM), and can also include a secondary memory 3710. Thesecondary memory 3710 can include, for example, a hard disk drive 3712and/or a removable storage drive 3714, representing a floppy disk drive,a magnetic tape drive, an optical disk drive, etc. The removable storagedrive 3714 reads from and/or writes to a removable storage unit 3716 ina well known manner. Removable storage unit 3716 represents a floppydisk, magnetic tape, optical disk, etc. which is read by and written toby removable storage drive 3714. As will be appreciated, the removablestorage unit 3716 includes a computer usable storage medium havingstored therein computer software and/or data.

In alternative embodiments, secondary memory 3710 may include othersimilar means for allowing computer programs or other instructions to beloaded into computer system 3702. Such means can include, for example, aremovable storage unit 3718 and an interface 3720. Examples of such caninclude a program cartridge and cartridge interface (such as that foundin video game devices), a removable memory chip (such as an EPROM, orPROM) and associated socket, and other removable storage units 3718 andinterfaces 3720 which allow software and data to be transferred from theremovable storage unit 3718 to computer system 3702.

Computer system 3702 can also include a communications interface 3722.Communications interface 3722 allows software and data to be transferredbetween computer system 3702 and external devices. Examples ofcommunications interface 3722 can include a modem, a network interface(such as an Ethernet card), a communications port, a PCMCIA slot andcard, etc. Software and data transferred via communications interface3722 are in the form of signals 3724 which can be electronic,electromagnetic, optical or other signals capable of being received bycommunications interface 3722. These signals 3724 are provided tocommunications interface via a channel 3726. This channel 3726 carriessignals 3724 and can be implemented using wire or cable, fiber optics, aphone line, a cellular phone link, an RF link and other communicationschannels. In the assay apparatus of the present invention, one exampleof channel 3726 is electrical connection 2902 that carries signal 3724of spectral emission 2918 to computer 2914.

In this document, the terms “computer program medium” and “computerusable medium” are used to generally refer to media such as removablestorage device, 3716 and 3718, a hard disk installed in hard disk drive3712, and signals 3724. These computer program products are means forproviding software to computer system 3702.

Computer programs (also called computer control logic) are stored inmain memory 3708 and/or secondary memory 3710. Computer programs canalso be received via communications interface 3722. Such computerprograms, when executed, enable the computer system 3702 to perform thefeatures of the present invention as discussed herein. In particular,the computer programs, when executed, enable the processor 3704 toperform the features of the present invention. Accordingly, suchcomputer programs represent controllers of the computer system 3702.

In an embodiment where the invention is implemented using software, thesoftware may be stored in a computer program product and loaded intocomputer system 3702 using removable storage drive 3714, hard drive 3712or communications interface 3722. The control logic (software), whenexecuted by the processor 3704, causes the processor 3704 to perform thefunctions of the invention as described herein.

In another embodiment, the invention is implemented primarily inhardware using, for example, hardware components such as applicationspecific integrated circuits (ASICs). Implementation of the hardwarestate machine so as to perform the functions described herein will beapparent to persons skilled in the relevant art(s).

In yet another embodiment, the invention is implemented using acombination of both hardware and software.

The assay apparatus of the present invention is particularly suited forcarrying out the methods of the present invention. To conduct amicroplate thermal shift assay using the method and apparatus of thepresent invention, samples are placed in a heat conducting block, heatedaccording to a predetermined temperature profile, stimulated with anexcitatory wavelength of light, and the spectral emission from thesamples is detected while the samples are being heated in accordancewith the predetermined temperature profile.

It is to be understood that the assay apparatus of the present inventionis not limited to use with the methods of the present invention orlimited to conducting assays on biological polymers, proteins, ornucleic acids. For example, the assay apparatus of the present inventioncan be used to incubate samples to a predetermined temperature.Alternatively, the assay apparatus of the present invention can be usedto perform polymerase chain reaction, thermal cycling steps for anypurpose, assaying thermal stability of a compound, such as a drug, todetermine conditions that stabilize a compound, or to determineconditions that facilitate crystallization of a compound.

Having now generally described the invention, the same will become morereadily understood by reference to the following specific examples whichare included herein for purposes of illustration only and are notintended to be limiting unless otherwise specified.

EXAMPLE 1 Ranking Ligands that Bind to the Active Site of Humanα-thrombin

Using the computer controlled process DirectedDiversity® (see U.S. Pat.No. 5,463,564), scientists at 3-Dimensional Pharmaceuticals, Inc. havegenerated a combinatorial library of compounds directed at the activesite of human α-thrombin. Approximately 400 compounds were synthesizedand assayed by a conventional spectrophotometric kinetic assay in whichsuccinyl-Ala-Ala-Pro-Arg-p-nitroanilide (Bachem, King of Prussia, Pa.)served as substrate. Five of these compounds, which are characterized byK_(i)'s that span almost four orders of magnitude in binding affinity,were used to test the range and limits of detection of the thermal shiftassay. These five proprietary compounds are listed in Table 3, alongwith the K_(i) for each respective compound, as measured by the kineticassay (last column). K_(i)'s for these compounds ranged from 7.7 nM for3dp-4026 to 20.0 μM for 3dp-3811.

A stock human α-thrombin solution (1.56 mg/mL) from Enzyme Research Labswas first diluted to 0.5 mg/mL (11 μM) with 50 mM Hepes, pH 7.5, 0.1 MNaCl (assay buffer, unless mentioned otherwise), and stored on ice. Thefive ligands (recrystallized solids characterized by mass spectrometryand NMR) were accurately weighed out to be 1.5 to 2.0 mg and dissolvedin 1.0 mL of 100% DMSO so that the concentration was between 1.8 and 3.8mM. A 96 well V-bottom Costar microplate was then set up such that 100μL of the 11 μM human α-thrombin solution was pipetted into wells A1through A6. This was followed by the addition of 2 μL of 3dp-3811 intowell A2, 2 μL of 3dp-3959 into well A3, 2 μL of 3dp-4077 into well A4, 2μL of 3dp-4076 into well A5, 2 μL of 3dp-4026 into well A6, and 2 μL of100% DMSO into control well A1. The contents were mixed by repeateduptake and discharge using a 100, μL pipette tip. Finally, one drop ofmineral oil (Sigma, St. Louis, Mo.) was added on top of the wells toreduce evaporation of samples at elevated temperatures. The microplatewas then placed on heating block 4 of a RoboCycler Gradient 96Temperature Cycler (Stratagene, La Jolla, Calif.), set at 25° C., for 1minute. The plate was then placed into a SPECTRAmax™ 250spectrophotometer (set to 30° C.) and the absorbance at 350 nm wasmeasured for each sample. This reading served as the blank or referencefrom which all the other readings at higher temperatures were compared.The assay was initiated by setting heating block 1 to 38° C.,programming the temperature cycler to move the microplate to heatingblock 1, and keeping the microplate there for 3 minutes. Following theequilibration at 38° C., the plate was moved to the 25° C. block (Block4) for 30 seconds, inserted in the spectrophotometer, and absorbance wasread at 350 nm. The microplate was then put back into the temperaturecycler and was moved to heating block 2, which had been pre-equilibratedat 40° C. After 3 minutes at 40° C., the plate was returned to 25° C.(on block 4) for 30 seconds, and was returned to the spectrophotometerfor a measurement of absorbance at 350 nm. This process was repeated 18more times until the temperature had been raised to 76° C. in 2° C.increments. After subtraction of the blank absorbance (A₃₅₀ at 25° C.),turbidity, reflected in the absorbance value, was plotted as a functionof temperature. The thermal denaturation curves for this experiment areshown in FIG. 1.

The control-(in well A1), which contained only 11 μM human α-thrombin in2% DMSO, was found to undergo a thermal denaturation transition startingat ˜50° C., as reflected in the large increase in A₃₅₀. The midpoint inthis transition was observed to be ˜55° C. This result was consistentwith differential scanning calorimetric measurements for bovineprothiombin 1, which revealed a denaturation transition at T_(m)=58° C.(Lentz, B. R. et al., Biochemistry 33:5460-5468 (1994)). The thermaldenaturation curves for all of the tested inhibitor compounds displayeda shift in the transition towards higher temperatures. 3dp-4026 showedthe largest shift in T_(m): ˜9° C. This result is consistent with thefact that, among the compounds tested, 3dp-4026 exhibited the greatestbinding affinity, as judged by kinetic measurements withsuccinyl-Ala-Ala-Pro-Arg-p-nitroanilide as substrate. Indeed, the rankorder of shifts in T_(m), shown in FIG. 1, paralleled the order ofbinding affinity as measured by conventional enzymology. These resultsindicate that by simply observing the shift in T_(m) for a series ofcompounds relative to the control, one can easily and correctly rank aseries of compounds in increasing order of binding affinity to theprotein of interest.

It was possible to take the microplate thermal shift assay one stepfurther and estimate the binding affinity of each ligand at T_(m). Thiswas done by substituting T₀, T_(m), ΔH_(u) and ΔC_(pu) into equation(1). If ΔH_(u) and ΔC_(pu) cannot be measured because a calorimetricdevice is not available, one can make educated guesses at ΔH_(u) andΔC_(pu) for the therapeutic target. In the case of human α-thrombin, itwas possible to use ΔH_(u)=200.0 kcal/mol, a value measured for theclosely related protein bovine prothrombin 1 (Lentz, B. R. et al.,Biochemistry 33:5460-5468 (1994)). A value of ΔC_(pu)=2.0 kcal/mol-° Kwas used to calculate K_(L) at T_(m) since similar proteins of this sizehave been shown to yield similar values. The binding affinities at T_(m)of the five test ligands closely paralleled the K_(i)'s measured with aspectrophotometric substrate (Table 3).

TABLE 3 Microplate Thermal Shift Assay for Ligands Binding to the ActiveSite of Human α-thrombin. Turbidity as an Experimental Signal. [Li-K_(d) at K_(i) Protein/ gand] T_(m) ΔT_(m) K_(d) at T_(m) ^(a) 310°K.^(b) (310° K.)^(c) Ligand (μM) (° K.) (° K.) (nM) (nM) (nM) Thrombinnone 327.15 0.0 (TH) TH/3dp- 37 328.15 1.0 14400 5880 2000 3811 TH/3dp-76 332.15 5.0 660 224 250 3959 TH/3dp- 48 333.15 6.0 160 51.7 46 4077TH/3dp- 60 334.15 7.0 76.3 23.6 26 4076 TH/3dp- 67 336.15 9.0 12.3 3.57.7 4026 ^(a)Calculations for K_(d) at T_(m) were made using equation(1) with ΔH^(T0) _(u) = 200.0 kcal/mole, as observed for prothrombin 1by Lentz, B. R. et al., Biochemistry 33:5460-5468 (1994), and anestimated ΔC_(pu) - 2.0 kcal/mole - ° K.; and K_(d) = 1/K_(a).^(b)Estimates for K_(d) at T = 310° K. were made using equation (3),where ΔH^(T) _(L) was estimated to be −10.0 kcal/mole. ^(c)K_(i) wasmeasured by classical enzymological methods that look at the [inhibotor]dependence of the enzymatic hydrolysis of the spectrophotometricsubstrate succinyl-Ala-Ala-Pro-Arg-p-nitroanilide at 310° K. (50 mMHepes, pH 7.5, 0.2 M NaCl, 0.05% β-octylglucoside).

EXAMPLE 2 Ranking Ligands that Bind to the Heparin Binding Site of Humanα-thrombin

Assays for ligands that bind to the heparin binding site of humanα-thrombin are more difficult to perform than assays for ligands thatbind to the active site of human α-thrombin. At the heparin bindingsite, no substrate is hydrolyzed, so no spectrophotometric signal can beamplified for instrumental detection. Heparin activity is usuallyestimated in biological clotting time assays. Alternatively, heparinbinding affinity for human α-thrombin can be determined by laboriouslyconducting 15 to 20 single point assays, in which the concentration oflow MW heparin is varied over two logs, and monitoring the quenching ofthe fluorescent probe, p-aminobenzamidine, bound to the active site ofhuman α-thrombin (Olson, S. T. et al., J Biol. Chem. 266:6342-6352(1991)). Thus, heparin binding to human α-thrombin represents the kindof challenge encountered with the vast majority of non-enzymereceptor/ligand binding events, which are commonly observed forhormone/receptor interactions, repressor/DNA interactions,neurotransmitter/receptor interactions, etc. Several heparin-likesulfated oligosaccharides and sulfated naphthalene compounds wereassayed by the microplate thermal shift assay. Using the thermal shiftassay, it was possible to use a single compound per well to quickly rankthe compounds in order of increasing binding affinity, with K_(d)'sranging over three orders of magnitude (see Table 4). Like theexperiment in Example 1, the thermal shift assay results agreed closelywith the results obtained through an alternative method, which requireda series of laborious (15 to 20 single determinations) fluorescencequench assays over a wide range of concentrations of low MW heparin(Olson, S. T. et al., J. Biol. Chem. 266:6342-6352 (1991)). Theseresults confirm that by simply observing the shift in T_(m) for a seriesof compounds, relative to the control, one can easily and correctly ranka series of compounds in increasing order of binding affinity for theprotein of interest.

A search of the literature did not locate alternatively measured bindingresults for the other ligands, which may attest to the difficulty ofthese experiments. However, the literature did reveal that pentosanpolysulfate PSO₄) (Sigma, St. Louis, Mo.), dextran SO₄ (Sigma, St. LouisMo.), and suramin (CalBiochem, LaJolla, Calif.) have been observed tohave anticoagulant properties. Indeed, pentosan polysulfate and suraminwere tested previously in clinical trials for anti-angiogenic activity,but were discounted due to toxic effects, many of which were describedas coagulation anomalies (Pluda, J. M. et al., J. Natl. Cancer Inst.85:1585-1592 (1993); Stein, C. A., Cancer Res. 53:2239-2249 (1993)). Theaffinities of pentosan PSO₄ and suramin at T_(m), as measured by thethermal shift assay, were found to be 7-fold and 5700-fold higher,respectively, than the affinity of heparin 5000 (Table 4). These resultssuggested that these ligands may alter clotting rates by interferingwith the heparin mediated binding of human α-thrombin to anti-thrombinIII (AT III), a protein co-factor for human α-thrombin activity.

The results in Table 4 revealed another advantage of the microplatethermal shift assay for screening compound libraries: the process isblind and unbiased in the sense that it detects ligand bindingregardless of whether it is at the active site, an allosteric cofactorbinding site, or at a protein subunit interface. The ability to detectligands that bind with high affinity to sites outside an enzyme's activesite will greatly facilitate discovery of lead molecules.

TABLE 4 Microplate Thermal Shift Assay for Ligands Binding to theHeparin Binding Site of Human α-thrombin. Turbidity as an ExperimentalSignal. K_(i) K_(d) at (298° K.) [Li- K_(d) 298° K.^(b) (nM) Protein/gand] T_(m) ΔT_(m) at T_(m) ^(a) (nM) Litera- Ligand (μm) (° K.) (° K.)(nM) Observed ture Thrombin (TH) none 329.15 0.0 TH/Heparan 61 329.650.5 38,300 7,570 — SO₄ TH/Heparin 50 330.15 1.0 19,700 3,810 — 3000TH/Heparin 44 330.15 1.0 17,200 3,490 5,400^(c) 5000 TH/Pentosan 40332.15 3.0 2,425 427 — PSO₄ TH/Dextran 48 336.15 7.0 68.8 10.1 — SO₄TH/Suramin 102 340.15 11.0 3.02 0.37 — ^(a)Calculations for K_(d) atT_(m) were made using equation (1) with ΔH^(T0) _(u) = 200.0 kcal/mole,as observed for pre-thrombin 1 by Lentz, B. R. et al., Biochemistry33:5460-5468 (1994), and an estimated ΔC_(pu) = 2.0 kcal/mole - ° K.;and K_(d) = 1/K_(a). The thrombin, human α-thrombin (Factor IIa), fromEnzyme Research Labs (South Bend, IN) was diluted to 0.5 mg/mL (11 #μM)using 50 mM Hepes, pH 7.5, 0.1 M NaCl (3-fold dilution). All ligandswere dissolved in the same buffer. ^(b)Estimates for K_(d) at T = 298°K. were made using equation (3), where ΔH^(T) _(L) is estimated to be−10.0 kcal/mole. ^(c)Olson, S. T. et al., J. Biol. Chem. 266:6342-6352(1991).

EXAMPLE 3 Ranking αFGF Ligands

The second therapeutic receptor tested in the microplate thermal shiftassay was acidic fibroblast growth factor (aFGF), a growth factor thatplays a key role in angiogenesis (Folkman, J. et al., J. Biol. Chem.267:10931-10934 (1992)). A synthetic gene for this protein was purchasedfrom R&D Systems (Minneapolis, Minn.), and was cloned and expressed inE. coli using methods similar to those described for basic fibroblastgrowth factor (bFGF) (Thompson, L. D. et al., Biochemistry 33:3831-3840(1994); Pantoliano, M. W. et al., Biochemistry 33:10229-10248 (1994);Springer, B. A. et al., J. Biol. Chem. 269:26879-26884 (1994)).Recombinant aFGF was then purified by heparin-sepharose affinitychromatography as described (Thompson, L. D. et al., Biochemistry33:3831-3840 (1994)). aFGF is also known to bind heparin/heparan, whichis a cofactor for mitogenic activity. Heparin-like molecules, such aspentosan PSO₄ and suramin, inhibit the growth factor's biologicalactivity. A microplate thermal assay of these compounds was set up in away similar to that described above for human α-thrombin. The change inturbidity, as a function of temperature, for each of the ligandssuramin, heparin 5000, and pentosan PSO₄, is shown in FIG. 2. Theresults are summarized in Table 5. The affinity constants covered afairly broad range of binding affinities, with pentosan PSO₄ showing thehighest affinity. The order of ligand binding affinity of pentosan PSO₄,heparin 5000 and suramin paralleled that found for bFGF, as measuredusing isothermal titrating calorimetry (Pantoliano, M. W. et al.,Biochemistry 33:10229-10248 (1994)). The lack of alternatively measuredbinding affinities for these compounds probably attests to thedifficulty of making these measurements using assays which do notmonitor physical, temperature-dependent changes.

The results in Table 5 are consistent with the results in Tables 3 and4. Simply observing the shift in T_(m) for a series of compoundsrelative to the control, one can easily and correctly rank a series ofcompounds in increasing order of binding affinity to the protein ofinterest.

TABLE 5 Microplate Thermal Shift Assay for Ligands Binding to aFGF.Turbidity as an Experimental Signal. K_(d) at K_(i) 298° K.^(b) (298°K.)^(c) Protein/ [Ligand] T_(m) ΔT_(m) K_(d) at T_(m) ^(a) (nM) (nM)Ligand (μM) (° K.) (° K.) (nM) Observed Literature aFGF none 317.15 0.0aFGF/EEEEE 50 317.15 0.0 >50,000 — aFGF/Dermatan SO₄ 50 318.15 1.037,000 12,700 — aFGF/EEEEEEEE 50 322.15 5.0 10,076 3,040 — aFGF/β-CD 14SO₄ 47 329.15 12.0 1055 213 1500 aFGF/suramin 200 330.15 13.0 3220 622aFGF/Heparin 5000 50 331.15 14.0 576 106  470 aFGF/Heparan SO₄ 61 333.1516.0 357 60 — aFGF/Pentosan PSO₄ 100 336.15 19.0 208 31  88^(a)Calculations for K_(d) at T_(m) were made using equation (1) with anestimated ΔH^(T0) _(u) = 60.0 kcal/mole, and an estimated ΔC_(pu) = 0.95kcal/mole - ° K.; and K_(d) = 1/K_(a). All ligands, except β-CD 14 SO₄,were purchased from Sigma and used without further purification. β-CD 14SO₄ was purchased from American Maize Products Co. (Hammond, IN). TheaFGF was diluted to 0.25 mg/mL in 50 mM Hepes, #pH 7.5, 0.1 M NaCl. Allligands were dissolved in the same buffer. ^(b)Estimates for K_(d) at T= 298° K. were made using equation (3), where ΔH^(T) _(L) is estimatedto be −10.0 kcal/mole. ^(c)No published binding affinity data for theseligands was found in the literature, but the affinities for theseligands, binding to bFGF, as measured by isothermal titratingcalorimetry, are shown (Thompson, L. D. et al., Biochemistry33:3831-3840 (1994); Pantoliano, M. W. et al., Biochemistry33:10229-10248 (1994)).

EXAMPLE 4 Ranking bFGF Ligands

The microplate thermal shift assay was used to assess ligands forbinding to the heparin binding site of basic fibroblast growth factor(bFGF). The gene for bFGF was purchased from R&D Systems and was clonedand expressed in E. coli as previously described (Thompson, L. D. etal., Biochemistry 33:3831-3840 (1994); Pantoliano, M. W. et al.,Biochemistry 33:10229-10248 (1994); Springer, B. A. et al., J. Biol.Chem. 269:26879-26884 (1994)). It was found that pentosan PSO₄ andsuramin bound to bFGF with binding affinities of 55 nM and 3.5 μM,respectively. This result for PSO₄ compared very well with the affinityof 88 nM observed for PSO₄ binding to bFGF, as determined by isothermaltitrating calorimetry.

EXAMPLE 5 Ranking Human α-Thrombin Ligands Using Fluorescence Emission

Because fluorescence measurements are more sensitive than absorbancemeasurements, a fluorescence thermal shift assay was used to assessligand binding to human α-thrombin. The fluorescence emission spectra ofmany fluorophores are sensitive to the polarity of their surroundingenvironment and therefore are effective probes of phase transitions forproteins (i.e., from the native to the unfolded phase). The most studiedexample of these environment dependent fluorophores is8-anilinonaphthalene-1-sulfonate (1,8-ANS), for which it has beenobserved that the emission spectrum shifts to shorter wavelengths (blueshifts) as the solvent polarity decreases. These blue shifts are usuallyaccompanied by an increase in the fluorescence quantum yield of thefluorophore. In the case of ANS, the quantum yield is 0.002 in water andincreases to 0.4 when ANS is bound to serum albumin.

ANS was used as a fluorescence probe molecule to monitor proteindenaturation. In the fluorescence assay, the final concentration ofhuman α-thrombin was 0.5 μM, which is 20-fold more dilute than theconcentrations used in the turbidity assays. This concentration of humanα-thrombin is in the range used for the kinetic screening assays.

ANS was excited with light at a wavelength of 360 nm. The fluorescenceemission was measured at 460 nm using a CytoFluor II fluorescencemicroplate reader (PerSeptive Biosystems, Framingham, Mass.). Thetemperature was ramped up as described above for the turbidity assays(see Example 1). The plot of fluorescence as a function of temperatureis shown in FIG. 3 for human α-thrombin alone, and for the3dp-4026/human α-thrombin complex. The denaturation transition for humanα-thrombin was clearly observed at 57° C., a temperature which is onlyslightly higher than that observed in the turbidity experiment. Theresult from the fluorescence assay is, nonetheless, in close agreementwith the T_(m) of 58° C. observed for prothrombin 1 from differentialscanning calorimetry experiments. Importantly, 3dp-4026 (at 67 μM) wasfound to shift the denaturation transition to ˜66° C. to give a shift inT_(m) of 9° C., which is identical to that found using turbidity as thedetection signal (Table 3).

The results in FIG. 3 and Table 4 illustrate several important points.First, at least a 20-fold increase in sensitivity can be gained byswitching from an absorbance to a fluorescence emission detectionsystem. This can be critical for those receptor proteins for whichsupplies are limited.

Second, in the fluorescence assays, the denaturation transition signalis much cleaner than the signal in the turbidity assays. In theturbidity assays, higher concentrations of protein led to precipitationof denatured protein. Precipitated protein contributed to the noisysignal.

Third, shifts in T_(m) measurements from the microplate thermal shiftassays are reproducible from one detection system to another.

EXAMPLE 6 Ranking Ligands To The D(II) Domain of FGFR1

The microplate thermal-shift assay was employed to test the binding ofheparin 5000 and pentosan PSO₄ to the known heparin binding site in theD(II) domain of fibroblast growth factor receptor 1 (FGFR1). D(II) FGFR1is a 124 residue domain which is responsible for most of the free energyof binding for bFGF. D(II) FGFR1 was cloned and expressed in E. coli.Recombinant D(II) FGFR1 was renatured from inclusion bodies essentiallyas described (Wetmore, D. R. et al., Proc. Soc. Mtg., San Diego, Calif.(1994)), except that a hexa-histidine tag was included at the N-terminusto facilitate recovery by affinity chromatography on a Ni²⁺ chelatecolumn (Janknecht, R. et al., Natl. Acad. Sci. USA 88:8972-8976 (1991)).D(II) FGFR1 was further purified on a heparin-sepharose column (Kan, M.et al., Science 259:1918-1921 (1993); Pantoliano, M. W. et al.,Biochemistry 33:10229-10248 (1994)). Purity was >95%, as judged bySDS-PAGE. The D(II) FGFR1 protein was concentrated to 12 mg/mL (˜1 mM)and stored at 4° C.

The D(II) FGFR1 protein was dissolved in an ANS solution to aconcentration of 1.0 mg/mL (70 μM). The quantum yield for ANS bound tothe denatured form of D(II) FGFR1 was lower than the quantum yield forANS bound to human α-thrombin Because ANS fluorescence is veryenvironment dependent (see Lakowicz, I. R, Principles of FluorescenceSpectroscopy, Plenum Press, New York (1983)), the quantum yield observedfor the denaturation of different proteins will vary. For D(II) FGFR1,the signal for the turbidity version of the assay, however, was nearlyundetectable. Despite the decreased sensitivity for D(II) FGFR1, ANSrescued this system for the microplate assay. A similar result wasobtained for Factor Xa, except that the fluorescence quantum yield forANS bound to denatured Factor Xa was almost as good as it was for humanα-thrombin. It was found that the fluorescence quantum yield for ANSbound to denatured bFGF was as high as the quantum yield for ANS bindingto human α-thrombin.

The results of D(II) FGFR1 binding experiments, as determined by themicroplate thermal shift assay, are shown in FIG. 4 and Table 6. As waspreviously demonstrated for all of the other receptor proteins describedabove, the microplate thermal shift assay facilitated the ranking ofligand binding affinities for D(II) FGFR1.

TABLE 6 Microplate Thermal Shift Assay for Ligands Binding to D(II)FGFR1. Fluorescence Emission as an Experimental Signal. K_(d) at K_(d)[Li- 298° K.^(b) (298° K.)^(c) Protein/ gand] T_(m) ΔT_(m) K_(d) atT_(m) ^(a) (μM) (μM) Ligand (μm) (° K.) (° K.) (μM) Observed LiteratureD(II) FGFR1 none 312.8 0.0 D(II) 150 317.9 5.1 30.0 13.6 85.3 FGFR1/Heparin 5000 D(II) 156 319.4 6.6 19.1 4.9 10.9 FGFR1/ Pentosan PSO₄^(a)Calculations for K_(d) at T_(m) were made using equation (1) with anestimated ΔH^(T0) _(u) = 60.0 kcal/mol, and an estimated ΔC_(pu) = 0.95kcal/mole - ° K.; and K_(d) = 1/k_(a) . The D(II) FGFR1 was diluted to1.0 mg/mL (70 μM) in 50 mM Hepes, pH 7.5, 0.1 M NaCl with 136 μM ANSpresent. All ligands were dissolved in the same buffer and diluted50-fold into the protein solution. ^(b)Estimates for K_(d) at T = 298°K. were made using equation (3), where ΔH^(T) _(L) = −12.1, and −7.48kcal/mole for the pentosan PSO₄ and heparin 5000, respectively, asdetermined by isothermal titrating calorimetry (Pantoliano, M. W. etal., Biochemistry 33:10229-10248 (1994)). ^(c)Published binding affinitydata for these ligands binding to D(II)-D(III) FGFR1 as determined bytitrating calorimetry (Pantoliano, M. W. et al., Biochemistry33:10229-10248 (1994)).

EXAMPLE 7 Microplate Thermal Shift Assay of Factor D

In order to further demonstrate the cross target utility of themicroplate thermal shift assay, another enzyme, Factor D, was tested forits ability to undergo thermal unfolding transitions. Factor D is anessential serine protease involved in the activation of the alternativepathway of the complement system, the major effector system of the hostdefense against invading pathogens. Factor D was purified from the urineof a patient with Fanconi's syndrome (Narayana et al, J. Mol. Biol.235:695-708 (1994)) and diluted to 4 μM in assay buffer (50 mM Hepes, pH7.5, 0.1 M NaCl). The assay volume was 10 μL and the concentration of1,8-ANS was 100 μM. The experiment was carried out using 15 μL roundbottom dimple plates (an 8×12 well array). The protein was heated in twodegree increments between 42° C. to 62° C., using a Robocycler™temperature cycler. After each heating step, and prior to fluorescencescanning using the CytoFluor II™ fluorescence plate reader the samplewas cooled to 25° C. (see Example 1). The non-linear least squares curvefitting and other data analysis were performed as described for FIG. 3.The results of the microplate thermal shift assay of Factor D is shownin FIG. 5 and reveal a thermal unfolding transition that occurs near 324K (51° C.) for the unliganded form of the protein. No reversible ligandsof significant affinity are known for Factor D. The results in FIG. 5show that the microplate thermal shift assay can be used to screen alibrary of compounds for Factor D ligands. The results in FIG. 5 alsoshow that the microplate thermal shift assay is generally applicable toany target molecule.

EXAMPLE 8 Microplate Thermal Shift Assay of Factor Xa

Human Factor Xa, a key enzyme in the blood clotting coagulation pathway,was chosen as yet another test of the cross target utility of themicroplate thermal shift assay. Factor Xa was purchased from Enzymeresearch Labs (South Bend, Ind.) and diluted 1.4 μM in assay buffer (50mM Hepes, pH 7.5, 0.1 M NaCl). The assay volume was 100 μL and theconcentration of 1,8-ANS was 100 μM. The protein was heated in twodegree increments between 50° C. to 80° C. using a Robocycler™temperature cycler. After each heating step, prior to fluorescencescanning using the CytoFluor II™ fluorescence plate reader, the samplewas cooled to 25° C. (see Example 1). The results of a microplatethermal shift assay of Factor Xa is shown in FIG. 6. A thermal unfoldingtransition was observed at 338 K (65° C.). Data analysis was describedas described for FIG. 3. The results in FIG. 6 show that the microplatethermal shift assay of protein stability is generally applicable to anytarget molecule.

EXAMPLE 9 Miniaturization of the Microplate Thermal Shift Assay ofLigands Binding to Human α-Thrombin

A miniaturized form of the microplate thermal shift assay was developedto minimize the amount of valuable therapeutic protein and ligandsrequired for the assay. In the first attempt at decreasing the assayvolume, the assay volume was decreased from 100 μL to 50 μL withoutadversely affecting the fluorescent signal. When the assay volume wasreduced further by a factor often, to 5 μL, favorable results wereobtained for human α-thrombin As shown in FIG. 7, the human α-thrombinunfolding transition could be easily observed at its usual T_(m). Moreimportantly, an active site inhibitor was observed to shift the T_(m) ofthe unfolding transition by 8.3° K to yield an estimate of the K_(d) of15 nM at the T_(m). The K_(a) at T_(m) was calculated using therelationship: $\begin{matrix}{K_{L}^{T_{m}} = \frac{\begin{matrix}{\exp \left\{ {{- {\frac{\Delta \quad H_{u}^{T_{0}}}{R}\left\lbrack {\frac{1}{T_{m}} - \frac{1}{T_{0}}} \right\rbrack}} +} \right.} \\\left. {\frac{\Delta \quad C_{pu}}{R}\left\lbrack {{\ln \left( \frac{T_{m}}{T_{0}} \right)} + \frac{T_{0}}{T_{m}} - 1} \right\rbrack} \right\}\end{matrix}}{\left\lbrack L_{T_{m}} \right\rbrack}} & \left( {{equation}\quad 1} \right)\end{matrix}$

where

K_(L) ^(T) ^(_(m)) =K_(a) at T_(m) (ligand associate constant at T_(m))

T_(m)=332.2° K (midpoint of the unfolding transition in the absence of aligand)

T₀=323.9° K

ΔH_(u) ^(T) ^(_(o)) =200.0 kcal/mol (enthalpy of unfolding for prethrombin observed by Lentz et al., 1994)

ΔC_(pu)=2.0 kcal/mol (estimated change in heat capacity of unfolding forhuman α-thrombin)

L_(T)=50.0 μM

The Kd at temperatures near 25 or 37° C. will be of higher affinity ifthe enthalpy of binding, ΔH_(b), is negative for this ligand. Using aspectrophotometric assay, an apparent K_(i) of approximately 8 nM wasobserved at 37° C. (310° K).

The measurements shown in FIG. 7 were obtained using the CytoFluor IIfluorescence plate reader (PerSeptive Biosystems, Framingham, Mass.). Inthe experiment, the excitation wavelength of light was 360 nm and theemission was measured at 460 nm. The microplates employed for thisminiaturized assay were either the conventional polycarbonate V-bottom96 well plate (Stratagene, or Costar) or polycarbonate plates thatcontain 15 μL dimples in an 8×12 array (Costar plate lids). In thereaction, the concentration of human α-thrombin was μM in assay buffer(50 mM Hepes, pH 7.5, 0.1 M NaCl). The assay volume was 5 μL and theconcentration of 1,8-ANS was 100 M The protein was heated in two degreeincrements between 14° C. to 64° C. using a Robocycler™ temperaturecycler. After each heating step, and prior to fluorescence scanningusing the CytoFluor II™ fluorescence plate reader the sample was cooledto 25° C. for 30 seconds (see Example 1). The non-linear least squarescurve fitting and other data analysis were performed as described forFIG. 3.

EXAMPLE 10 Miniaturization of the Microplate Thermal Shift Assay ofLigands Binding to D(II) FGFR1

Recombinant D(II) FGFR1 was purified from inclusion bodies and purifiedby affinity chromatography on heparin sepharose. A stock solution ofD(II) FGFR1 (15 mg/mL; 1.1 mM) was diluted to 50 μM in assay buffer (50mM Hepes, pH 7.5, 0.1 M NaCl). The assay volume was 10 μL and theconcentration of 1,8-ANS was 250 μM. The unfolding transition in theabsence of ligands was found to be about 312 K (39° C.) as shown in FIG.8. In the presence of the heparin mimic aprosulate (300 μM), theunfoding transition was observed to increase by about 8 K to about 320K. Using this temperature midpoint T_(m), it is possible to estimate thebinding affinity of aprosulate to D(II)FGFR1 to be about 18 μM at theT_(m) (Table 6). These results demonstrate the ability of the microplatethermal shift assay to estimate ligand binding affinity to a non-enzymetarget molecule.

EXAMPLE 11 Miniaturization of the Microplate Thermal Shift Assay ofUrokinase

Another target molecule analyzed was human urokinase-type plasminogenactivator (u-Pa.). U-PA enzymatically converts plasminogen into theactive protease plasmin. U-PA is involved in tissue remodeling, cellularmigration and metastases. The gene for u-PA was obtained from ATCC(Rockville, Md.) and modified to appropriately express active enzyme inE. coli. u-PA was cloned, overexpressed in E. coli, and purified usingprocedures similar to those described by Winkler et al. (Biochemistry25:4041-4045 (1986)). The last step of u-PA purification was performedin the presence of the active site inhibitorglu-gly-arg-chloromethylketone (CMK) and hence the u-PA utilized forthis experiment was the CMK-u-PA complex. The experiment was performedin the miniaturized format in 5 μL well volume. One μL of concentratedCMK-u-PA g/L, 371.4 g/L, 371.4 μM was added to 4 μL of 62.5 mM MOPS, pH7, 125 mM NaCl, and 250 μM 1,8-ANS, in multiple wells of a 96-wellpolycarbonate V-bottom microtiter plate. A thermal denaturation curvewas generated as previously described for thrombin, aFGF, D(II)FGFR1,Factor D, and Factor Xa, by incremental heating of the microplatefollowed by a fluorescence reading after each temperature increase.Analysis and non-linear least squares fitting of the data for thisexperiment show that the T_(m) for CMK-u-PA under these conditions is81° C., which is considerably higher Man that seen for thrombin, aFGF,D(II)FGFR1, Factor D, and Factor Xa (55,44,40, 51, 55, and 65° C.,respectively). This experiment demonstrates the utility of the currentinvention in determining the T_(m) for relatively thermostable proteinsor proteins stabilized by the high affinity binding of ligand(s) andfirer demonstrates the ability to perform such an experiment in aminiaturized format

EXAMPLE 12 Further Miniaturization of the Microplate Thermal Shift Assayof Human α-thrombin

A stock thrombin solution was diluted to 1 μM in 50 mM Hepes, pH 7.5,0.1 M NaCl and 100 M 1,8-ANS. An electronic multi-channel pipettor wasused to dispense either 2 μL or 5 μL of diluted thrombin solution intowells of a 96-well polycarbonate microtiter plat. The plate wassubjected to 3 minutes of heating in a thermal block capable ofestablishing a temperature gradient across the microplate, followed by30 seconds cooling to 25° C., and subsequent reading in the CytoFluor IIfluorescence plate reader. Data were analyzed by non-linear leastsquares fitting and plotted as shown in FIGS. 10 and 11. Each curverepresents a replicate experiment. Standard deviations for T_(m)determinations were very good for experiments utilizing either 5 μL or 2μL volumes (+/−1.73 and +/−0.90 K, respectively), demonstrating theability of the current invention to operate at very low volumes. Infact, the volume which one could employ in the current invention seemsto be limited only by the technology available to dispense small volumesaccurately.

The assay volume was reduced to 2 μL, as shown for human α-thrombin (1.0μM) in FIG. 11. Reproducible pipetting of 2 μL in a 96 well arrayrequires the employment of specialized pipetting tools such as themulti-channel pipettor available from Matrix Technologies Corp. (Lowell,Mass.) which has ±2.0% or 0.15 μL precision and ±2.5% or 0.15 μLaccuracy for volumes 0.5 to 12.5 μL.

EXAMPLE 13 Single Temperature Mode of the Microplate Thermal Shift Assay

Results of a single temperature assay are shown in FIG. 12. Thecompounds 3DP-3811, 3DP-3959, 3DP4076, and 3DP4660 bind to the activesite of human α-thrombin The K_(i)'s (enzymatically determined) of thesefour compounds for human α-thrombin are of 20,000 nM, 250 nM, 25 nM, and8 nM, respectively. Each of these four compounds were equilibrated withhuman α-thrombin in separate 5 μl assay volumes in a 96 well plate. Thefinal ligand concentration was 50 μM.

For the ligands that bind to human α-thrombin with higher affinity, lowlevels of fluorescence emission were observed, relative to the controlreaction (human α-thrombin alone) at 55° C. The result for the samplecontaining the weakly binding ligand 3DP-3811 was little different fromthe result obtained for the control sample. The decrease in fluorescenceemission for 3DP4076 was not as large as expected, given its highaffinity (K_(i) of 25 nM for human α-thrombin. This result could be duein part to the lower solubility of chloride salts of this compound.

The data in FIG. 12 clearly demonstrate the utility of the singletemperature embodiment of the microplate thermal shift assay for quicklyidentifying ligands with binding affinities (K_(d)'s) of 250 nM orbetter when the ligand concentration is 50 μM.

EXAMPLE 14 Microplate Thermal Shift Assay of Intrinsic ProteinTryptophan Fluorescence Emission

The intrinsic Trp fluorescence of human α-thrombin was assayed in amicroplate thermal shift assay. 100 μL samples contained 2 μM humanα-thrombin. The samples were exposed to light from a Xenon-Arc lamp at280 nm. Emission was detected at 350 nm using the BioLumin 960(Molecular Dynamics). Temperature cycling, between 14° C. and 66° C.,was performed as described in previous examples. The results of theassay are shown in FIGS. 13 and 14. A small increase in fluorescenceemission was observed at 350 nm with increasing temperature. However,this increase in fluorescence emission was barely detectable above thelevel of fluorescence in the blank wells that contained no protein (FIG.13). Subtracting an average blank improved the signal to noise ratio(FIG. 14), but the observed unfolding transition was different from thattypically observed in assays employing 1,8-ANS. In contrast to thetransition observed using 1,8-ANS, the transition in FIG. 14 appearsbroader and has a midpoint temperature T_(m) at 334.4±5.1° K, some fivedegrees higher than the T_(m) observed for human α-thrombin in assaysperformed with 1,8-ANS.

EXAMPLE 15 Assay of Multi-Ligand Binding Interactions

As previously demonstrated, the thermal shift assay can be used for thescreening of ligands for binding to single sites on target proteins. Inlight of the underlying physical principles upon which the microplatethermal shift assay is based, the near additivity of the free energy ofligand binding and protein unfolding, it is possible to employ themicroplate thermal shift assay for analyzing multi-ligand bindinginteractions with a target protein. If the free energy of binding ofdifferent ligands binding to the same protein are nearly additive, thenone can analyze multi-ligand binding systems, whether the ligands bindin a cooperative (positive) fashion or a non-cooperative (negative)fashion.

Multiple ligand binding to human α-thrombin was assayed in a microplatethermal shift assay. Human α-thrombin it has at least four differentligand binding sites: (1) the catalytic binding site; (2) the fibrinbinding site (exosite I); (3) the heparin binding site (exosite II); and(4 ) the Na⁺ binding site, located ˜15 Å from the catalytic site. First,independent binding of three individual ligands was assayed: 3DP-4660,Hirugen (hirudin 53-64) (Bachem), and heparin 5000 (CalBiochem). Theseligands bind to the catalytic site, the fibrin binding site and theheparin binding site, respectively.

A stock thrombin solution was diluted to 1 μM in 50 mM Hepes, pH 7.5,0.1 M NaCl, 1 mM CaCl. and 100 μM 1,8-ANS. Each thrombin ligand wasincluded singly and in various combinations to 1 μM thrombin solutionsat final concentrations of 50 μM each, except for heparin 5000, whichwas 200 μM. 100 μL of thrombin or thrombin/ligand(s) solution wasdispensed into wells of a 96-well V-bottom polycarbonate microtiterplate. The plate was subjected to 3 minutes of heating in a thermalblock capable of establishing a temperature gradient across themicroplate, followed by 30 seconds cooling at 25° C., and subsequentreading in a fluorescence plate reader. Data were analyzed by non-linearleast squares fitting.

The results of these individual binding reactions are shown in FIGS. 15and 16. The rank order of binding affinity was 3DP4660>Hirugen>heparin5000, corresponding to K_(d) values of 15 nM, 185 nM and 3434 nM,respectively, for the ligands binding at each T_(m) (see Equation (4 )).

The results reveal thermal unfolding shifts that are slightly smallerthan would be expected if the free energies of binding were fullyadditive. For example, Hirugen alone displays a ΔT_(m) of 5.8° C., and3DP4660 alone displays a ΔT_(m) of 7.7° C. In combination, however,Hirugen and 3DP4660 display a ΔT_(m) of 12.2° C. This result means thatthe binding affinity of one or both ligands is diminished when bothligands are bound, and is an example of negative cooperativity inbinding between the fibrin and catalytic binding sites. Such anegatively cooperative effect is consistent with the human α-thrombinliterature, in which the kinetics of hydrolysis of various chromogenicsubstrates were found to depend upon ligands binding to exosite I.Indeed, a 60% decrease in K_(m) for the hydrolysis ofD-phenylalanylpipecolyl arginyl-p-nitroanilide was observed when Hirugenwas present (Dennis et al., Eur. J Biochem. 188:61-66 (1990)). Moreover,there is also structural evidence for cooperativity between thecatalytic site and exosite I. A comparison of the isomorphous structuresof human α-thrombin bound to PPACK (a human α-thrombin catalytic siteinhibitor) and Hirugen revealed conformational changes that occur at theactive site as a result of Hirugen binding at the exosite I(Vijayalakshmi et al, Protein Science 3:2254-2271 (1994)). Thus, in themicroplate thermal shift assay, the apparent cooperativity observedbetween the catalytic center and the exosite I is consistent withfunctional and structural data in the literature.

Similarly, when the binding of all three ligands was assayed, a ΔT_(m)of 12.9° C. was observed (FIG. 16). If the free energies of binding werefully additive, one would expect to observe a ΔT_(m) of 17.7° C. Theobserved result means that flier negative cooperativity occurs vialigand binding at all three protein binding sites. This result isconsistent with the literature. In a ternary complex with heparin andfibrin monomer, human α-thrombin has decreased activity towardtri-peptide chromogenic substrates and pro-thrombin (Hogg & Jackson, J.Biol. Chem. 265:248-255 (1990)), and markedly reduced reactivity withanti-thrombin (Hogg & Jackson, Proc. Natl. Acad. Sci. USA 86:3619-3623(1989)). Also, recent observations indicate that ternary complexes alsoform in plasma and markedly compromise heparin anticoagulant activity(Hotchkiss et al., Blood 84:498-503 (1994)). A summary of thesemulti-ligand binding results is shown in Table 7.

The results in FIG. 15, FIG. 16, Table 7 illustrate the followingadvantages of using the microplate thermal shift assay to performmulti-variable analyses. First, the same microplate thermal shift assaycan be used to simultaneously detect the binding of multiple ligands atmultiple binding sites in a target protein. Second, the microplatethermal shift assay can be used to detect the same ligand binding to twoor more sites in a therapeutic target. Third, the microplate thermalshift assay affords the detection of cooperativity in ligand binding.Information about ligand binding cooperativity can be collected andanalyzed very quickly. Thus, multi-ligand binding experiments that wouldtake months to perform using alternative technologies take only hours toperform using the microplate thermal shift assay.

TABLE 7 Microplate thermal shift assay for Ligands Binding to the ActiveSite, Exosite, and Heparin Binding Site of Human α-thrombin K_(d) at[Ligand] T_(m) ΔT_(m) K_(d) at T_(m) ^(a) 298°^(b) Protein/Ligand (μM)(° K.) (° K.) (nM) (nM) Thrombin (TH) none 323.75 0.0 TH/Heparin 5000200 327.95 4.2 3434 470 TH/Hinidin 53-65 50 329.52 5.8 185 23TH/3dp-4660 50 331.40 7.7 29 3 TH/Heparin 5000 200 327.95 TH/Hep./Hir.50 330.57 2.6 4254 478 TH/Heparin 5000 200 327.95 TH/Hep.3dp 4660 50333.20 5.3 350 32 TH/Hirudin 53-65 50 329.52 TH/Hir./Hep. 200 330.57 1.175422 8467 TH/Hirudin 53-65 50 329.52 TH/Hir.3dp-4660 50 335.97 6.5 1179 TH/3dp-4660 50 331.40 TH/3dp-4660/Hep 200 333.20 1.8 38205 351TH/3dp-4660 50 331.40 TH/3dp-4660/Hir. 50 335.97 4.6 731 54^(a)Calculations for K_(d) at T_(m) were made using equation (1) withΔH_(u)^(T° = 200.0 kcal/mole, as observed for pre-thrombin 1 by Lentz et al., (1994), and an estimated ΔC)_(pu) = 2.0 kcal/mole-° K.; and K_(d) = 1/K_(a). ^(b)Estimates for K_(d)at T = 298° K. were made using the equation (3), where ΔH_(L)^(T is estimated to be −10.0 kcal/mole.)

EXAMPLE 16 Screening Biochemical Conditions that Increase Humanα-thrombin Stability

The microplate thermal shift assay was used, with four differentfluorophores, to simultaneously screen the effects of multiple pHvalues, sodium chloride concentrations, and reduction-oxidationcompounds on human α-thrombin stability. Thrombin solution was dilutedto 1 μM in 50 mM Hepes, pH 7.5, NaCl at either 0.1 M or 0.5 M, 10 mMEDTA, 10 mM CaCl₂, 10 mM dithiothreitol, 10 1 mM CaCl₂, and 100 μM1,8-ANS, 10% (v/v) glycerol, or 0.1% (w/v) polyethylene glycol (PEG)6000. Reaction volume was 100 μL.

The results of these multi-variable experiments are shown in FIGS. 17A-Dand FIG. 18. FIGS. 17A-D summarize the stability data collected in asingle 96 well plate for human α-thrombin. In FIG. 17A, the fluorophoreis 1,8-ANS. In FIG. 17B, the fluorophore is 2,6-ANS. In FIG. 17C, thefluorophore is 2,6-TNS. In FIG. 17D, the fluorophore is bis-ANS. Theresults in FIGS. 17A-D show a pH optimum of about 7.0 and an increase instability with increasing NaCl concentration. A ΔT_(m) of about 12° C.was observed when the NaCl concentration was increased from 0 to 0.5 M.FIG. 18 shows a stabilizing effect of 10% glycerol and a destabilizingeffect of dithiothreitol. From FIGS. 17A-D and 18 is evident that theflourophores 1,8-ANS and 2,6-TNS are most effective in the microplatethermal shift assay.

The stabilizing effect of NaCl is particularly interesting since thereare recent reports in the literature of a weak Na⁺ binding site (K_(d)of 30±3 mM in 5 mM Tris buffer pH 8.0, 0.1% PEG, 25° C.) approximately15 Å from the catalytic center of thrombin (Dang et al., NatureBiotechnology 15:146-149 (1997)). Using equation (1), it is possible toestimate the NaCl binding to be ˜6 mM near the T_(m) (53° C.) in 50 mMHepes pH 8.0 buffer (zero and 0.10 M NaCl).

The additional stabilization that occurs at a NaCl concentration ofgreater than 0.10 M may come from additional Na⁺ and/or Cl⁻ bindingevents summed over the entire structure of human α-thrombin.Alternatively, the source of this further stabilization may come fromless specific salting out effect that is usually observed at 0.5 to 2 MNaCl and is due to the preferential hydration of proteins induced bysalts (Timasheff & Arakawa, In: Protein Structure, A Practical Approach,T. E. Creighton, ed., IRL Press, Oxford, UK (1989), pp. 331-354)).

The stabilizing effect of glycerol on proteins has been attributed to abalance between the preferential exclusion of glycerol (i.e.preferential hydration of proteins) and the specific binding to polarregions on the surface of proteins (Timasheff & Arkawa, In: ProteinStructure, A Practical Approach, T. E. Creighton, ed., IRL Press,Oxford, UK (1989), pp. 331-354)).

EXAMPLE 17 Screening Biochemical Conditions that Increase D(II) FGFReceptor 1 Stability

The microplate thermal shift assay was used to simultaneously screen theeffects of multiple biochemical conditions on D(II) FGF receptor 1stability. The assays were performed by mixing 1 μL of D(II) FGFR1 (froma 500 μM concentrated stock in 50 mM HEPES pH 7.5) with 4 μL of eachbiochemical condition in wells of a 96-well polycarbonate microtiterplate. Final protein concentration after mixing was 100 μM and final1,8-ANS concentration was 200 μM. Biochemical conditions were tested asfollows: The pH's tested were 5 (Ma acetate), 6 (MES), 7 (MOPS), 8(HEPES), and 9 (CHES), with final buffer concentrations of 50 mM.

The salt concentrations tested were 0.1 or 0.5 M NaCl. Additives weretested in 50 mM MOPS, pH 7, 0.1 M NaCl, at final concentrations of 1 mM(EDTA, dithiothreitol), 10 mM (CaCl₂, MgCl₂, MgSO₄, NiSO₄), 50 mM(arginine), 100 mM ((NH₄)₂SO₄, LiSO₄, Na₂SO₄, ZnSO₄), 5% w/v(polyethylene glycol 6000), and 10% v/v glycerol.

Thermal denaturation profiles were generated as previously described forthrombin, aFGF, Factor D, and Factor Xa, by incremental heating of themicroplate followed by a fluorescence reading after each temperatureincrease. Data were analyzed by non-linear least squares fitting asdescribed previously.

The results of these multi-variable experiments are shown in FIGS.19-24. As shown in FIG. 19, stability increased with increasing NaClconcentration. ΔT_(m) of about 5° C. was observed as NaCl concentrationwas increased from 0.1 to 0.5 M. As shown in FIG. 20, both MgSO₄ andarginine stabilized the protein. As shown in FIG. 21, 10% glycerolstabilized the protein. Further, salts of the Hofmeister series such asLi₂SO₄, Na₂SO₄, (NH₄)₂SO₄ and Mg₂SO₄ all had stabilizing effects (FIG.21). As shown in FIG. 22, dithiothreitol destabilllzed the protein.These results are not very different form that of human α-thrombin. Asshown in FIG. 23, a pH optimum of about 8.0 was observed. The relativestabilizing effects of EDTA, CaCl₂, MgCl₂, MgSO₄, arginine, (NH₄)₂SO₄,Li₂, SO₄, Na₂SO₄, glycerol, polyethylene glycol 6000, and dithiothreitolare shown in FIG. 24.

EXAMPLE 18 Screening Biochemical Conditions that Increase UrokinaseStability

The microplate thermal shift assay was used to simultaneously screen theeffects of multiple biochemical conditions on human urokinase stability.This experiment was performed by mixing 1 μL of urokinase (from a 371 5Mconcentrated stock in 20 mM Tris pH 8) with 4 μL of each biochemicalcondition in wells of a 96-well polycarbonate microtiter plate. Finalprotein concentration after mixing was 74 μM and final 1,8-ANSconcentration was 200 μM. Biochemical conditions were tested as follows:The pH's tested were 5 (acetate), 6 (MES), 7 (MOPS), 8 (HEPES), and 9(CHES) with final buffer concentrations of 50 mM. The saltconcentrations tested were 0.1 or 0.5 M NaCl. Glycerol was tested at 10%v/v in 50 mM MOPS, pH 7, 0.1 M NaCl.

Thermal denaturation profiles were generated as previously described forthrombin, aFGF, Factor D, D(II) FGFR1, and Factor Xa, by incrementalheating of the microplate followed by a fluorescence reading after eachtemperature increase. Data were analyzed by non-linear least squaresfitting as described previously.

The results of these multi-variable experiments are shown in FIG. 25. ApH optimum of about 7.0 was observed. Increasing concentrations ofsodium chloride stabilized the protein. 10% glycerol also stabilized theprotein. These results are consistent with the results reported in theliterature (Timasheff & Arakawa, In: Protein Structure, A PracticalApproach, T. E. Creighton, ed., IRL Press, Oxford, UK (1989), pp.331-354).

FIGS. 17-25 illustrate the advantage of using the microplate thermalshift assay to simultaneously screen for multi-variable biochemicalconditions that optimize protein stability. Using the methods andapparatus of the present invention, one can rapidly screen large arraysof biochemical conditions for conditions that influence the stability ofproteins. Thus, the present invention can be used to rapidly identifybiochemical conditions that optimize protein shelf-life.

EXAMPLE 19 Screening Biochemical Conditions that Facilitate ProteinFolding

Factorial experiments were performed to identify biochemical conditionsthat increased the yield of correctly folded His₆-D(II)-FGFR1.His₆-D(II)-FGFR1 is recombinant D(II) FGF receptor 1 protein, to which apolyhistidine tag is attached to the N-terminus. The results aresummarized in Table 8. When the final guanidinium hydrochlorideconcentration was 0.38 M, a refolded protein yield of 13.5±0.2% wasobtained at pH 8.0 and 0.5 M NaCl. This yield could be increased to15.5±0.3% if glycerol was present at 7% (v/v). A further increase inHis₆-D(II)-FGFRI refolding yield to about 18% was observed when the pHwas increased to 8.9. In fact, increasing the pH from 8.0 to 8.9improved the yields in all experiments. These results demonstrate that apH between 8 and 9, and 7% glycerol, are two important conditions thatfacilitate D(II)-FGFRI folding. Each of these conditions increased thefolded protein yield by about 15 to 20% over the starting conditions atpH 8.0 and 0.5 M NaCl.

Importantly, the effects of pH and glycerol appear to be nearlyadditive. The increased yield of refolded protein at pH 8.9 and 7%glycerol was found to be 17.8%, 32% higher than the yield obtained at apH 8.0 and 0.5 M NaCl (13.5±0.2% yield). The near additivity ofrefolding determinants has important consequences since it suggests thatthe small individual free energy components that comprise the overallfree energy of folding can be incrementally combined to optimize theyield of folded protein.

TABLE 8 Factorial Experiment to Optimize the Protein Folding Yield forImmobilized His₆D(II)-FGFR1 at a final Gdn-HCl concentration of0.38M^(a) 7% Glycerol/ 500 mM NaCl 50 mM NaCl 50 mM NaCl pH 8.013.3%^(b)  9.3% 15.1% pH 8.0 13.6%  9.4% 15.8% pH 8.9 16.1% 13.5% 17.8%pH 8.9 10.3% 17.8% ^(a)Refolding was initiated by diluting a 3.2 mLsuspension of Ni²⁺NTA/6M Gdn-HCl to 50 mL in the respective refoldingbuffers (1:15.6 dilution) so that the final Gdn-HCl concentration was0.38M. ^(b)Yields are based on measured A₂₈₀ values for fractions elutedoff a Heparin Sepharose column. The immobilized protein concentrationwas 1.2 mg/mL, as measured by a Bio-Rad protein assay. Since the columnsize was 21 mL, 25.2 mg of D(II) FGFR1 was bound to the resin.

Results of a second round of refolding experiments at a final Gdn-HClconcentration of 0.09 M revealed that the Gdn-HCl is an even moreimportant factor affecting the folding of His₆-D(II)FGFR1 (Table 9). AtpH 8.0 and 0.5 M NaCl, decreasing the Gdn-HCl concentration to 0.09 Mdoubled the refolded protein yield, relative to the yield obtained at pH8.0, 0.5 M NaCl, and 0.38 M Gdn-HCl (Table 9). In accordance with theresults obtained at a Gdn-HCl concentration of 0.38 M, the yield ofrefolded His₆-D(II)-FGFR1 in 0.09 M Gdn-HCl was also increased in thepresence of glycerol. These results suggest that the improved yield ofrefolded His₆D(II)-FGFR1 in glycerol (5 to 10%) and lower Gdn-HClconcentration are additive. Further, the results in Table 9 reveal thatthe Hofmeister salt Na₂SO₄ increases the yield of refolded proteinalmost as well as 5 to 10% glycerol.

TABLE 9 Factorial Experiment to Optimize the Protein Folding Yield forImmobilized His₆D(II)-FGFR1. Final Gdn-HCl of 0.09M^(a) 5% 10% GlycerolGlycerol 100 300 500 mM 50 mM 50 mM 50 mM mM mM NaCl NaCl NaCl NaClNa₂SO₄ Na₂SO₄ pH 8.0 25.6%^(b) 29.7% 36.5% 35.6% 32.2% 33.4%^(a)Refolding was initiated by diluting a 7.5 mL suspension ofNi²⁺NTA/6M Gdn-HCl to 50 mL in the respective refolding buffers (1:6.7dilution) so that the final Gdn-HCl concentration was 0.09M. ^(b)Yieldsare based on measured A₂₈₀ values for fractions eluted off a HeparinSepharose column. The immobilized protein concentration was 1.6 mg/mL,as measured by Bio-Rad protein assay. Since the column size was 20 mL,32 mg of D(II) FGFR1 was bound to the resin.

Upon comparison of the biochemical conditions that increase the yield ofrefolded Ni²⁺NTA bound His6-D(II)-FGFR1 (Tables 8 and 9) and thoseconditions that increase the overall protein stability ofHis₆-D(II)-FGFR1 (FIGS. 19-24), it is clear that there is a strongcorrelation between the protein folding results and the proteinstability results. Glycerol, salts of the Hofmeister series, and pH 8.5to 8.9 improve protein folding yield and overall protein stability ofHis₆-D(II)-FGFR1.

These results are consistent with the model of protein folding in FIG.26. If the aggregation of unfolded His₆-D(II)-FGFR1 is suppressed whenimmobilized to Ni²⁺NTA, and a simple two state equilibrium existsbetween U and N, then the factors that influence the relative positionof the equilibrium between U and N should be the same whether one startsfrom U (in the refolding experiment) or start from N (in the microplatethermal shift assay protein stability screen). Since thermodynamics arepath independent, only the initial and final states of this reactionshould be important. Since similar biochemical conditions facilitateprotein stability and folded protein yield, the simple model for proteinfolding depicted in FIG. 26 is accurate for this protein. Thus, themicroplate thermal shift assay can serve as a rapid and general methodfor screening biochemical conditions that optimize protein folding.

EXAMPLE 20

FIG. 28 shows the results of microplate thermal shift assays of usingeach of four fluorescence probe molecules: bis-ANS, 2,6-TNS, 1,8-TNS,and 2,6-ANS. Thrombin solution was diluted to 1 μM in 50 mM Hepes, pH7.5, and 0.1 M NaCl.

EXAMPLE 21 Comparison of Assay Results for a Fluorescence Scanner and aCharge Coupled Device Camera

A Gel Documentation and Analysis System (Alpha Innotech Corp., SanLeandro, Calif.) was used to perform a microplate thermal shift assay.This system uses a CCD camera to detect fluorescence emission fromstained gels, dot blot assays, and 96 well plates. The excitatory lightsource was a long wavelength UV tans-illumination box located directlybelow the CCD camera. The 96 well plate to be assayed was placed on thetrans-illumination box within the focal viewing area of the CCD camera(21×26 cm).

A 2 μM solution of human α-thrombin was prepared in 50 mM Hepes, pH 7.5,0.1 M NaCl by diluting a 34 μM stock solution (1:17) of purified humanα-thrombin (Enzyme Research Labs, Madison, Wis.). The human α-thrombinsolution also contained 100 μM 1,8-ANS. 100 μL of the humanα-thrombin-1,8-ANS solution was aliquoted into each of twelve wells of asingle row (row A) of a V-bottom polycarbonate microplate (Costar). Agradient block (RoboCycler™, Stratagene) was used to heat the twelvesamples, from 14 to 66° C., across the rows of the microplate. i.e. atemperature gradient of 2° C. per well was established. Thus, well Alwas at 66° C. and well A12 was at 14° C. The control solution thatcontained 100 μM 1,8 ANS in the same buffer (no protein) was placed ineach of wells B1 to B12. After adding a drop of mineral oil to each wellto prevent evaporation, the plate was heated on the gradient block for 3min. The contents of each well were then allowed to reach roomtemperature and transferred to a flat bottom microplate. In thisexperiment, no filters were employed to narrow the excitatory wavelengthto ˜360 nm and the emission wavelength to ˜460 nm, which are optimalwavelengths for the 1,8 ANS fluorophore. The flat bottom plate was thenplaced on the near UV transillumination box and the CCD camera was usedto measure the amount of emitted light. The plate was also read using aconventional fluorescence plate reader (CytoFluor II), in order tocompare the results obtained by the two different detection methods. Theresults for the two detection methods are plotted in FIG. 40. Theresults in FIG. 40 show that the CCD camera is useful as a fluorescenceemission detector for monitoring the unfolding of a protein in themicroplate thermal shift assay.

EXAMPLE 22 Microplate Thermal Shift Assay Using a Charge Coupled DeviceCamera

An emission filter was used to block out all stray light outside theregion of the emission region for 1,8-ANS (˜460 nm). In addition, the 5μL miniaturized form of the microplate thermal shift assay was employedto test the CCD camera detection method in this configuration. Both thepolycarbonate V-bottom and dimple plates were tested. The experiment wasessentially the same as described in Example 21, except that the volumeof the assay was 5 μL in either the V-bottom or dimple 96 well plates.The temperature range was 14 to 66° C. (right to left) for the V-bottomplate, and 46 to 70° C. (right to left) for the dimple plate.Photographs of the CCD images are shown in FIG. 41. The V-bottom wellmicroplate image is shown in FIG. 41A. The dimple plate image is shownin FIG. 41B. The results obtained from the plate in FIG. 41A is shown inFIG. 42. The results in FIG. 42 show that data obtained using a CCDcamera compare very well with data obtained using a fluorescence platereader that employs a photo-multiplier tube (PMT) for fluorescencedetection.

All publications and patents mentioned hereinabove are herebyincorporated in their entireties by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

What is claimed is:
 1. In a combinatorial method for generating leadcompounds which comprises synthesizing a multiplicity of compounds andtesting the ability of said compounds to bind to a receptor molecule,the improvement comprising (a) contacting said receptor molecule withone compound of said multiplicity of compounds in each of a multiplicityof wells in a microplate; (b) simultaneously heating said multiplicityof wells from step (a); (c) measuring in each of said wells a physicalchange associated with the thermal unfolding of said receptor moleculeresulting from said heating; (d) generating a thermal unfolding curvefor said receptor molecule as a function of temperature for each of saidwells; (e) comparing each of said thermal unfolding curves in step (d)with the thermal unfolding curve obtained for said receptor molecule inthe absence of any of said compounds in said multiplicity of differentcompounds; and (f) ranking the affinities of said multiplicity ofdifferent compounds for said receptor molecule according to the changein each of said thermal unfolding curves.
 2. The method of claim 1,wherein said unfolding is denaturing, and wherein said thermal unfoldingcurve is a thermal denaturation curve.
 3. The method of claim 1, whereinsaid step (d) further comprises determining a midpoint temperature(T_(m)) from the thermal unfolding curve; wherein said step (e)comprises (e1) comparing the T_(m) of each of said unfolding curves instep (d) to (i) the T_(m) of each of said other thermal unfolding curvesand to (ii) the T_(m) of the thermal unfolding curve obtained for saidreceptor molecule in the absence of any of said compounds in saidmultiplicity of different compounds; and wherein said step (f) comprises(f1) ranking the efficacies of said multiplicity of different compoundsfor said receptor molecule according to the change in T_(m) of each ofsaid thermal unfolding curves.
 4. The method of claim 1, wherein saidstep (c) comprises measuring the absorbance of light by said contents ofeach of said multiplicity of wells.
 5. The method of claim 1, whereinsaid receptor molecule is a protein.
 6. The method of claim 5, whereinsaid step (a) comprises contacting said receptor molecule with saidmultiplicity of different compounds, in the presence of a fluorescenceprobe molecule present in each of said multiplicity of wells, andwherein said step (c) comprises (c1) exciting said fluorescence probemolecule, in each of said multiplicity of wells, with light; and (c2)measuring the fluorescence from each of said multiplicity of wells. 7.The method of claim 6, wherein said fluorescence is fluorescenceemission.
 8. The method of claim 6, wherein said fluorescence probemolecule is a DAPOXYL™ derivative.
 9. The method of claim 6, whereinsaid step (c2) further comprises measuring the fluorescence from a eachof said multiplicity of wells simultaneously.
 10. The method of claim 5,wherein said step (c) comprises (c1) exciting tryptophan residues insaid protein, in each of said multiplicity of wells, with light; and(c2) measuring the fluorescence from each of said multiplicity of wells.11. The method of claim 1 wherein said receptor molecule is a nucleicacid.
 12. The method of claim 11, wherein said step (c) comprisesmeasuring the change in hyperchromicity of said nucleic acid.
 13. Themethod of claim 11, wherein said receptor molecule is a fluorescentlylabeled double-stranded oligonucleotide, wherein one strand of saidoligonucleotide contains a donor fluorophore and the other strand ofsaid oligonucleotide contains an acceptor fluorophore, wherein said step(a) comprises contacting said oligonucleotide with said multiplicity ofdifferent molecule in each of said multiplicity of wells, and whereinsaid step (c) comprises (c1) exciting said donor fluorophore, in each ofsaid multiplicity of wells, with light; and (c2) measuring thefluorescence from said acceptor fluorophore in each of said multiplicityof wells.
 14. The method of claim 1, further comprising selecting one ormore compounds from said multiplicity of wells by their maximal bindingaffinity for said receptor molecule and generating a second library ofcompounds having high affinity for said receptor molecule.
 15. Themethod of claim 1, wherein said multiplicity of compounds comprises acombinatorial library.
 16. The method of claim 14, wherein saidcombinatorial library is a directed diversity chemical library.